Fundamentals of Biological System

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LESSON - 1

CELL

CONTENTS

1.0 Aims and Objectives

1.1 Introduction

1.2 Structure of Prokaryotic and Eukaryotic Cell

1.3 Let Us Sum Up

1.4 Points for Discussion

1.5 Check your Progress

1.6 Lesson – End Activities

1.7 References

1.0 AIMS AND OBJECTIVESTo know about the cell and its types, etc

A cell is a microscopic, structural and functional unit of living organismscapable of independent existence (e.g. Amoeba). All living things are composedof cells. Some functioning cells come together to form a tissue and tissuescollectively form organs. In more complex living organisms, organs worktogether for the purpose of survival as system. However, in all living organisms,the cell is a functional unit and all of biology revolves around the activity ofthe cell.

The study of cell is impossible without the microscope. The first simplemicroscope was prepared by Anton Van Leewenhoek (1632-1723) who studiedthe structure of bacteria, protozoa, spermatozoa, red blood cells etc. The word‘cell’ was first coined by Robert Hooke in 1665 to designate the empty honey-comb like structures viewed in a thin section of bottle cork which he examined.He was impressed by the microscopic compartments in the cork as theyreminded him of rooms in a monastery which are known as cells. He thereforereferred to the units as cells. In 1838, the German botanist Matthios Schleidenproposed that all the plants are made up of plant cells. Then in 1839, hiscolleague, the anatomist Theodore Schwann studied and concluded that allanimals are also composed of animal cells. Schwann and Schleiden studied awide variety of plant and animal tissues and proposed the “cell theory” in1839. It stated that “all organisms are composed of cells.” But still the realnature of a cell was in doubt. Cell theory was again rewritten by Rudolf

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Virchow in 1858 and said that all living things are made up of cells and thatall cells arise from pre-existing cells. It was German biologist Schulze whofound in 1861 that the cells are not empty as were seen by Hooke but containa ‘stuff’ of life called protoplasm. During the 1950s scientists developed theconcept that all organisms may be classified as prokaryotes or eukaryotes.For example, in prokaryotic cells, there is no nucleus; eukaryotic cells have anucleus. Another important difference between prokaryotes and eukaryotesis that the prokaryotic cell does not have any

1.1. INTRODUCTIONIntracellular components. Bacteria and blue- green algae come under

the prokaryotic group, and

1.2 STRUCTURE OF PROKARYOTIC AND EUKARYOTIC CELLCells in our world come in two basic types, prokaryotic and eukaryotic.

“Karyose” comes from a Greek word which means “kernel,” as in a kernel ofgrain. In biology, we use this word root to refer to the nucleus of a cell. “Pro”means “before,” and “eu” means “true,” or “good.” So “Prokaryotic” means“before a nucleus,” and “eukaryotic” means “possessing a true nucleus.” Thisis a big hint about one of the differences between these two cell types.Prokaryotic cells have no nuclei, while eukaryotic cells do have true

Fig. 1. : Prokaryotic and Eukaryotic Cell

Nuclei: Despite their apparent differences, these two cell types have a lotin common. They perform most of the same kinds of functions, and in thesame ways. Both are enclosed by plasma membranes, filled with cytoplasm,and loaded with small structures called ribosome. Both have DNA whichcarries the archived instructions for operating the cell. And the similaritiesgo far beyond the visibility; for example, the DNA in the two cell types isprecisely the same kind of DNA, and the genetic code for a prokaryotic cell isexactly the same genetic code used in eukaryotic cells. The difference is thatthe prokaryotic cell has a cell wall which is absent in animal cells. However,many kinds of eukaryotic cells do have cell walls. The size and complexity doexist. Eukaryotic cells are much larger and much more complex than

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prokaryotic cells. These two observations are not unrelated to each other.

If we take a closer look at the comparison of these cells, we see thefollowing differences:

Eukaryotic cells have a true nucleus, bound by a double membrane.Prokaryotic cells have no nucleus. The purpose of the nucleus is to sequesterthe DNA-related functions of the big eukaryotic cell into a smaller chamber,for the purpose of increased efficiency. This function is unnecessary for theprokaryotic cell, because its much smaller size means that all materialswithin the cell are relatively close together. Of course, prokaryotic cells dohave DNA and DNA functions. Biologists describe the central region of thecell as its “nucleoid” (-oid=similar or imitating), because it’s

1. pretty much where the DNA is located. But note that the nucleoid isessentially an imaginary “structure.” There is no physical boundaryenclosing the nucleoid.

2. Eukaryotic DNA is linear complexed with proteins called “histones,”and is organized into chromosomes; prokaryotic DNA is “naked,”meaning that it has no histones associated with it, and it is notformed into chromosomes. Though many are sloppy about it, the term“chromosome” does not technically apply to anything in a prokaryoticcell. A eukaryotic cell contains a number of chromosomes; a prokaryoticcell contains only one circular DNA molecule (has no ends) and avaried assortment of much smaller circlets of DNA called “plasmids.”The smaller, simpler prokaryotic cell requires far fewer genes tooperate than the eukaryotic cell.

3. Both cell types have many ribosomes, but the ribosomes of theeukaryotic cells are larger and more complex than those of theprokaryotic cell. Ribosomes are made out of a special class of RNAmolecules (ribosomal RNA, or rRNA) and a specific collection ofdifferent proteins. A eukaryotic ribosome is composed of five kinds ofrRNA and about eighty kinds of proteins. Prokaryotic ribosomes arecomposed of only three kinds of rRNA and about fifty kinds of protein.

4. The cytoplasm of eukaryotic cells is filled with a large, complexcollection of organelles, many of them enclosed in their ownmembranes; the prokaryotic cell contains no membrane-boundorganelles which are independent of the plasma membrane. This is avery significant difference, and the source of the vast majority of thegreater complexity of the eukaryotic cell.

5. There is much more space within a eukaryotic cell than within aprokaryotic cell, and many of these structures, like the nucleus,increase the efficiency of functions by confining them within smallerspaces within the huge cell, or with communication and movementwithin the cell.

6. One aspect of that evolutionary connection is particularly interestingwithin eukaryotic cells by the presence of fascinating organelle called

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mitochondria. And in plant cells, an additional family of organellescalled plastids, the most famous of which is the renowned chloroplast.Mitochondria (the plural of mitochondrion) and chloroplasts almostcertainly have a similar evolutionary origin. Both are pretty clearlythe descendants of independent prokaryotic cells, which have takenup permanent residence within other cells through a well-knownand very common phenomenon called endosymbiosis.

One structure not shown in our prokaryotic cell is called a mesosome,which is an elaboration of the plasma membrane-a sort of rosette of ruffledmembrane intruding into the cell and not all prokaryotic cells have these.

Fig. 2. : Prokaryotic cell and Mitochondrion

Fig. 2 shows a trimmed down prokaryotic cell, including only the plasmamembrane and a couple of mesosomes. A mitochondrion is included forcomparison. The similarities in appearance between these structures arepretty clear. The mitochondrion is a double-membrane organelle, with a smoothouter membrane and an inner membrane which protrudes into the interior ofthe mitochondrion in folds called cristae. This membrane is very similar inappearance to the prokaryotic plasma membrane with its mesosomes, howevernot more significant than appearance. Both the mesosomes and the cristaeare used for the same function: the aerobic part of aerobic cellular respiration.

Fig. 3. : Prokaryotic and Eukaryotic cells

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Cellular respiration is the process by which a cell converts the raw, potentialenergy of food into biologically useful energy, and there are two general types,anaerobic (not using oxygen) and aerobic (requiring oxygen). In practical terms,the big difference between the two is that aerobic cellular respiration has amuch higher energy yield than anaerobic respiration. Aerobic respiration isclearly the evolutionary offspring of anaerobic respiration. (In anaerobicrespiration with additional chemical sequences added on to the end of theprocess to allow utilization of oxygen).

Protozoa, fungi, animals, and plants come under the eukaryotic group

1.3 LET US SUM UP* All living things are composed of cells.* The word ‘cell’ was first coined by Robert Hooke in 1665.* Cell is basically of two types prokaryotic and eukaryotic.

1.4 POINTS FOR DISCUSSION* “Cell in the basic unit of life” – Comment.

1.5 CHECK YOUR PROGRESS

WRITE DOWN THE MAIN FEATURES OF A EUKARYOTIC CELLNote: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer............................................................................................................................................................................................................................................................................................................................................................................................................................................................

1.6 LESION-END ACTIVITIES1) Define cell.2) Write down the main differences between prokaryotic and eukaryotic

cells

1.7 REFERENCES1. Lehinger, A.L. 1984, Principles of Biochemistry, CBS Publishers and

distributors, New Delhi, India.2. Horton, Moran, Ochs, Rawn, Scrimgeour Principles of Biochemistry,

Prentice Hall Publishers.3. Shanmughavel, P. 2005, Principles of Bioinformatics, Pointer

Publishers, Jaipur, India.4. David, E. Sadava Cell Biology: Organelle structure and Fucntion Jones

& Bartlett Publishers.

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LESSON - 2

CELL ORGANELLES AND THEIR FUNCTIONS

CONTENTS


2.1 Mitochondria

2.2 Chloroplasts

2.3 Endoplasmic reticulum

2.4 Golgi apparatus

2.5 Ribosome

2.6 Lysosome

2.7 Nucleus

2.8 Nucleolus

2.9 Peroxisome

2.10 Let Us Sum Up


2.12 Lesson-end activities


2.14 References

2.0 AIMS AND OBJECTIVESIn this lesson we’ve learn about the cell organelles and their functions.

A living cell is a complex, multi-functional unit. Even the simplest of cellsperforms a large array of different tasks and functions by the arrangement ofthe cell organelles such as cell wall and plasma membrane and cytosolicsubstances such as nucleus, Golgi bodies, endoplasmic reticulum,mitochondria etc.

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2.1 MITOCHONDRIA

Fig. 4. : Mitochondrial Components

Mitochondria are the cells’ power sources. They are distinct organelleswith two membranes. Usually they are rod-shaped, however they can be round.The outer membrane limits the organelle. The inner membrane is throwninto folds or shelves that project inward are called “cristae mitochondriales”.They contain two membranes, separated by a space. Both are the typical“unit membrane” (railroad track) in structure. Inside the space enclosed bythe inner membrane is the matrix. Which contains dense strands of DNA,ribosomes, or small granules and can code for part of their proteins withthese molecular tools.

The food we eat is oxidized to produce high-energy electrons that areconverted to stored energy. This energy is stored in high energy phosphatebonds in a molecule called adenosine triphosphate, or (ATP). Which is convertedfrom adenosine diphosphate by adding the phosphate group with the high-energy bond. Various reactions in the cell can either use energy (wherebythe ATP is converted back to ADP, releasing the high energy bond) or produceit (whereby the ATP is produced from ADP). Let us break down each of thesteps so you can see how food turns into ATP energy packets and water. Thefood we eat must first be converted to basic chemicals that the cell can use.Some of the best energy supplying foods contains sugars or carbohydrates.Using bread as an example, the sugars are broken down by enzymes that splitthem into the simplest form of sugar which is called glucose. Then, glucoseenters the cell by special molecules in the membrane called “glucosetransporters”.

Once inside the cell, glucose is broken down to make ATP in two pathways.The first pathway requires no oxygen and is called anaerobic metabolism.This pathway is called glycolysis and it occurs in the cytoplasm outside themitochondria. During glycolysis, glucose is broken down into pyruvate. Other

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foods like fats can also be broken down for use as fuel (see following cartoon).Each reaction is designed to produce some hydrogen ions (electrons) that canbe used to make energy packets (ATP). However, only 4 ATP molecules can bemade by one molecule of glucose run through this pathway. That is whymitochondria and oxygen are so important. We need to continue the breakdownprocess with the Kreb’s cycle inside the mitochondria in order to get enoughATP to run all the cell functions.

Fig. 5. : ATP Synthase

Pyruvate is carried into the mitochondria and there it is converted intoAcetyl Co-A which enters the Kreb’s cycle. This first reaction produces carbondioxide because it involves the removal of one carbon from the pyruvate

MITOCHONDRIAL MEMBRANE MORPHOLOGYThe outer membrane of the mitochondria contains the protein “porin”.

This forms an aqueous channel through which proteins up to 10,000 daltonscan pass and go into the intermembrane space. Indeed, the small moleculesactually equilibrate between the outer membrane and the cytosol. However,most proteins cannot get into the matrix unless they pass through the innermembrane. This membrane contains cardiolipin which renders it virtuallyimpermeable and requires transport mechanisms across the membrane thatare more organized and regulated.

Fig. 6. : Protein import by Mitochondria

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Transport across the mitochondrial membranes requires the concertedaction of a number of translocation machineries. The machinery in the outermembrane is called the Tom complex (Translocator outer membrane) andthat for the inner membrane is called the Tim complex (Translocator InnerMembrane). Proteins that have to go all the way to the matrix have an NH2cleavable signal sequence and become uncoiled or stretched out to go throughthe translocators. This involves ATP binding and is monitored and stabilizedby a chaperone protein, including hsp70. Thus, before the protein can gothrough Tom complex, it must become “translocation competent”, and processedas follows:

1. First, as with many mitochondrial proteins, Tom40 requires cytosolicchaperones to prepare it for entry. In the case of this protein, becoming“translocation competent” requires ATP and a partially folded state(the latter is mediated by the cytosolic chaperone (hsp70).

2. Second, when it is “competent”, it interacts with the surface receptor,Tom20. There is no cleavable signal peptide however, the experimentsshowing the requirement for partial folding suggests targetinginformation is found in discontinuous sites brought together in thefolded domain.

3. Final insertion is into preexisting Tom complexes and requires anintact N terminus.

4. Dimerization occurs after entry into the membrane.5. Tim54 carries a amino terminal, noncleaved translocation sequence

that is positively charged. However, it prefers to use Tom70 as itsreceptor instead of Tom20. After moving through the GIP, it uses itspositively charged amino terminal sequence to enter the matrix. Itrequired chaperones and ATP to get to the matrix.

6. Tim22 is a hydrophobic protein that uses Tom20 for targeting to theOM. Then it follows the Tim route for carrier proteins, like Tim23.and does not require hsp70 or ATP for entry.

7. Small Tims are normally found in the intermembrane space and arenot membrane proteins. They used Tom20 for their receptor andtransfer to the GIP complex. However, when Tom20 was destroyed bytrypsin, leaving only Tom5, the small Tims were able to enter.

2.2 CHLOROPLASTS

Chloroplasts are organelles found in plant cells and eukaryotic algae thatconduct photosynthesis. Chloroplasts absorb sunlight and use it in conjunctionwith water and carbon dioxide to produce sugars, the raw material for energyand biomass production in all green plants and the animals that depend onthem, directly or indirectly, for food. Chloroplasts capture light energy fromthe sun to conserve free energy in the form of ATP and reduce NADP toNADPH through a complex set of processes called photosynthesis. It is derivedfrom the Greek words chloros which means green and plast which means

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form or entity. Chloroplasts are members of a class of organelles known asplastids. They have their own genome, and may contain 60-100 genes.

STRUCTUREChloroplasts are observable morphologically as flat discs usually 2 to 10

micrometers in diameter and 1 micrometer thick. The chloroplast is containedby an envelope that consists of an inner and an outer phospholipid membrane.Between these two layers is the intermembrane space. The material withinthe chloroplast is called the stroma, corresponding to the cytosol of the originalbacterium, and contains one or more molecules of small circular DNA. It alsocontains ribosomes, although most of its proteins are encoded by genescontained in the host cell nucleus, with the protein products transported tothe chloroplast.

Within the stroma are stacks of thylakoids, the sub-organelles which arethe site of photosynthesis. The thylakoids are arranged in stacks called grana(singular: granum). A thylakoid has a flattened disk shape. Inside it is anempty area called the thylakoid space or lumen. Photosynthesis takes placeon the thylakoid membrane; as in mitochondrial oxidative phosphorylation, itinvolves the coupling of cross-membrane fluxes with biosynthesis via thedissipation of a proton electrochemical gradient. Embedded in the thylakoidmembrane is the antenna complex, which consists of proteins, and light-absorbing pigments, including chlorophyll and carotenoids. This complex bothincreases the surface area for light capture, and allows capture of photonswith a wider range of wavelengths. The energy of the incident photons isabsorbed by the pigments and funneled to the reaction centre of this complexthrough resonance energy transfer. Two chlorophyll molecules are thenionised, producing an excited electron which then passes onto thephotochemical reaction centre.

Chloroplast membrane: Chloroplasts contain several important membranes,vital for their function. Like mitochondria, chloroplasts have a double-membrane envelope, called the chloroplast envelope. Each membrane is aphospholipid bilayer, between 6 and 8 nm thick, and the two are separated bya gap of 10-20nm, called the intermembrane space. The outer membrane ispermeable to most ions and metabolites, but the inner membrane is highlyspecialised with transport proteins within the inner membrane, in the regioncalled the stroma, there is a system of interconnecting flattened membranecompartments, called the lamellae, or thylakoids. These are the sites of lightabsorption and ATP synthesis, and contain many proteins, including thoseinvolved in the electron transport chain. Photosynthetic pigments such aschlorophyll á and B, and some others e.g. xanthophylls and carotenoids arealso located within this space. The membranes of the chloroplasts containphotosystems I and II which harvest solar energy in order to excite electronswhich travel down the electron transport chain. and along the way is used topump H+ ions from the stroma into the thylakoid space. A concentration gradientis formed, which allows chemiosmosis to occur, where the protein ATP synthaseharvests the potential energy of the Hydrogen ions and uses it to combine

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ADP and a phosphate group to form ATP.

FUNCTIONSPhotosynthesis:

The heart of photosynthesis as it occurs in most autotrophs consists oftwo key processes:

* the removal of hydrogen (H) atoms from water molecules* the reduction of carbon dioxide (CO2) by these hydrogen atoms to

form organic molecules.

The second process involves a cyclic series of reactions named the CalvinCycle (after its discoverer). It is discussed in Photosynthesis: Pathway ofCarbon Fixation. The detail of the first process is our topic here.

The electrons (e”) and protons (H+) that make up hydrogen atoms arestripped away separately from water molecules.

2H2O -> 4e” + 4H+ + O2

The electrons serve two functions:* They reduce NADP+ to NADPH for use in the Calvin Cycle.* They set up an electrochemical charge that provides the energy for

pumping protons from the stroma of the chloroplast into the interiorof the thylakoid.

The protons also serve two functions:* They participate in the reduction of NADP+ to NADPH.* As they flow back out from the interior of the thylakoid (by facilitated

diffusion), passing down their concentration gradient), the energythey give up is harnessed to the conversion of ADP to ATP.

* Because it is drive by light, this process is called photophosphorylation.ADP + Pi -> ATP

The ATP provides the second essential ingredient for running the CalvinCycle.

The removal of electrons from water molecules and their transfer to NADP+

requires energy. The electrons are moving from a redox potential of about+0.82 volt in water to “0.32 volt in NADPH. Thus enough energy must beavailable to move them against a total potential of 1.14 volts. Where does theneeded energy come from? The answer: Light.

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Fig. 7. : Calvin Cycle

The Thylakoid Membrane

Chloroplasts contain a system of thylakoid membranes surrounded by afluid stroma.

Six different complexes of integral membrane proteins are embedded inthe thylakoid membrane. The exact structure of these complexes differs fromgroup to group (e.g., plant vs. alga) and even within a group (e.g., illuminatedin air or underwater). But, in general, one finds:

1. PHOTOSYSTEM IThe structure of photosystem I in a cyanobacterium (“blue-green alga”)

has been completely worked out. It probably closely resembles that of plantsas well.

It is a homotrimer with each subunit in the trimer containing:* 12 different protein molecules bound to* 96 molecules of chlorophyll a

o 2 molecules of the reaction center chlorophyll P700

o 4 accessory molecules closely associated with themo 90 molecules that serve as antenna pigments

* 22 carotenoid molecules* 4 lipid molecules

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* 3 clusters of Fe4S4

* 2 phylloquinones

2. PHOTOSYSTEM IIPhotosystem II is also a complex of* > 20 different protein molecules bound to* 50 or more chlorophyll a molecules

o 2 molecules of the reaction center chlorophyll P680

o 2 accessory molecules close to themo 2 molecules of pheophytin (chlorophyll without the Mg++)o the remaining molecules of chlorophyll a serve as antennapigments.

* some half dozen carotenoid molecules. These also serve as antennapigments.

* 2 molecules of plastoquinone

2.3 ENDOPLASMIC RETICULUM

The endoplasmic reticulum or ER is an organelle found in all eukaryoticcells that is an interconnected network of tubules, vesicles and cisternaethat is responsible for several specialized functions: Protein translation, folding,and transport of proteins to be used in the cell membrane (e.g., transmembranereceptors and other integral membrane proteins), or to be secreted (exocytosed)from the cell (e.g., digestive enzymes); sequestration of calcium; and productionand storage of glycogen, steroids, and other macromolecules. The endoplasmicreticulum is part of the endomembrane system. The basic structure andcomposition of the ER membrane is similar to the plasma membrane.

Structure: The general structure of the endoplasmic reticulum is anextensive membrane network of cisternae (sac-like structures) held togetherby the cytoskeleton. The phospholipid membrane encloses a space, the cisternalspace (or lumen), from the cytosol. The functions of the endoplasmic reticulumvary greatly depending on the exact type of endoplasmic reticulum and thetype of cell in which it resides. The three varieties are called roughendoplasmic reticulum, smooth endoplasmic reticulum, and sarcoplasmicreticulum.

Rough endoplasmic reticulum: The surface of the rough endoplasmicreticulum is studded with protein-manufacturing ribosomes giving it a “rough”appearance (hence its name). But it should be noted that these ribosomesare not resident of the endoplasmic reticulum incessantly. The ribosomesonly bind to the ER once it begins to synthesize a protein destined for sorting.The membrane of the rough endoplasmic reticulum is continuous with theouter layer of the nuclear envelope. Although there is no continuous membrane

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between the rough ER and the Golgi apparatus, membrane bound vesiclesshuttle proteins between these two compartments. The rough endoplasmicreticulum works in concert with the Golgi complex to target new proteins totheir proper destinations.

Smooth endoplasmic reticulum: Smooth endoplasmic reticulum is foundin a variety of cell types (both animal and plant) and it serves different functionsin each. The Smooth ER also contains the enzyme Glucose-6-phosphatasewhich converts Glucose-6-phosphate to Glucose, a step in gluconeogenesis.The Smooth ER consists of tubules and vesicles that branch forming a network.In some cells there are dilated areas like the sacs of rough endoplasmicreticulum. The network of smooth endoplasmic reticulum allows increasedsurface area for the action or storage of key enzymes and the products ofthese enzymes. The smooth endoplasmic reticulum is known for its storage ofcalcium ions in muscle cells. The smooth endoplasmic reticulum has functionsin several metabolic processes, including synthesis of lipids, metabolism ofcarbohydrates and calcium concentration, drug detoxification, and attachmentof receptors on cell membrane proteins. It is connected to the nuclear envelope.

Fig. 8. : Endoplasmic Reticulum

Sarcoplasmic reticulum: The sarcoplasmic reticulum is a special type ofsmooth ER found in smooth and striated muscle. The only structural differencebetween this organelle and the smooth endoplasmic reticulum is the medleyof protein they have, both bound to their membranes and drifting within theconfines of their lumens. This fundamental difference is indicative of theirfunctions: the smooth ER is built to synthesize molecules and the sarcoplasmicreticulum is built to store and pump calcium ions. The sarcoplasmic reticulumcontains large stores of calcium, which it sequesters and then releases whenthe cell is depolarised. This has the effect of triggering muscle contraction.

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FUNCTIONSThe endoplasmic reticulum serves many general functions, including the

facilitation of protein folding and the transport of synthesized proteins insacs called cisternae. Correct folding of newly-made proteins is made possibleby several endoplasmic reticulum chaperone proteins, including proteindisulfide isomerase (PDI), ERp29, the Hsp70 family member Grp78, calnexin,calreticulin, and the peptidylpropyl isomerase family. Only properly-foldedproteins are transported from the rough ER to the Golgi complex.

TRANSPORT OF PROTEINSSecretory proteins, mostly glycoproteins, are moved across the endoplasmic

reticulum membrane. Proteins that are transported by the endoplasmicreticulum and from there throughout the cell are marked with an addresstag called a signal sequence. The N-terminus (one end) of a polypeptide chain(i.e., a protein) contains a few amino acids that work as an address tag,which are removed when the polypeptide reaches its destination. Proteinsthat are destined for places outside the endoplasmic reticulum are packedinto transport vesicles and moved along the cytoskeleton toward theirdestination.

The endoplasmic reticulum is also part of a protein sorting pathway. It is,in essence, the transportation system of the eukaryotic cell. The majority ofendoplasmic reticulum resident proteins are retained in the endoplasmicreticulum through a retention motif. This motif is composed of four aminoacids at the end of the protein sequence. The most common retention sequenceis KDEL (lys-asp-glu-leu). However, variation on KDEL does occur and othersequences can also give rise to endoplasmic reticulum retention. It is notknown if such variation can lead to sub-endoplasmic reticulum localizations.There are three KDEL receptors in mammalian cells, and they have a veryhigh degree of sequence identity. The functional differences between thesereceptors remain to be established.

OTHER FUNCTIONS* Insertion of proteins into the endoplasmic reticulum membrane:

Integral proteins must be inserted into the endoplasmic reticulummembrane after they are synthesized. Insertion into the endoplasmicreticulum membrane requires the correct topogenic sequences.

* Glycosylation: Glycosylation involves the attachment ofoligosaccharides.

* Disulfide bond formation and rearrangement: Disulfide bonds stabilizethe tertiary and quaternary structure of many proteins.

Drug Detoxification: The smooth ER is the site at which some drugs aredetoxified.]

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2.4 GOLGI APPARATUS

The Golgi apparatus (also called the Golgi body, Golgi complex, ordictyosome) is an organelle found in typical eukaryotic cells. It was identifiedin 1898 by the Italian physician Camillo Golgi and was named after him. Theprimary function of the Golgi apparatus is to process and packagemacromolecules synthesised by the cell, primarily proteins and lipids. TheGolgi apparatus forms a part of the endomembrane system present ineukaryotic cells.

Structure: The Golgi is composed of membrane-bound sacs known ascisternae. Between five and eight are usually present, however as many assixty have been observed. Surrounding the main cisternae are a number ofspherical vesicles which have budded off from the cisternae. The cisternaestack has five functional regions: the cis-Golgi network, cis-Golgi, medial-Golgi, trans-Golgi, and trans-Golgi network. Vesicles from the endoplasmicreticulum (via the vesicular-tubular cluster) fuse with the cis-Golgi networkand subsequently progress through the stack to the trans-Golgi network,where they are packaged and sent to the required destination. Each regioncontains different enzymes which selectively modify the contents dependingon where they are destined to reside.

Fig. 9. : The Golgi Apparatus

FUNCTION

1. Cells synthesise a large number of different macromolecules requiredfor life. The Golgi apparatus is integral in modifying, sorting, and packagingthese substances for cell secretion (exocytosis) or for use within the cell. Itprimarily modifies proteins delivered from the rough endoplasmic reticulum,but is also involved in the transport of lipids around the cell, and the creationof lysosomes. In this respect it can be thought of as similar to a post office; itpackages and labels “items” and then sends them to different parts of thecell.

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2. Enzymes within the cisternae are able to modify substances by theaddition of carbohydrates (glycosylation) and phosphate (phosphorylation) tothem. In order to do so the Golgi transports substances such as nucleotidesugars into the organelle from the cytosol. Proteins are also labelled with asignal sequence of molecules which determine their final destination. Forexample, the Golgi apparatus adds a mannose-6-phosphate label to proteinsdestined for lysosomes. The Golgi also plays an important role in the synthesisof proteoglycans, molecules present in the extracellular matrix of animals,and it is a major site of carbohydrate synthesis.

3. This includes the productions of glycosaminoglycans or GAGs, longunbranched polysaccharides which the Golgi then attaches to a proteinsynthesized in the endoplasmic reticulum to form the proteoglycan. Enzymesin the Golgi will polymerize several of these GAGs via a xylose link onto thecore protein. Another task of the Golgi involves the sulfation of certainmolecules passing through its lumen via sulphotranferases that gain theirsulphur molecule from a donor called PAPs. This process occurs on the GAGsof proteoglycans as well as on the core protein. The level of sulfation is veryimportant to the proteoglycans signalling abilities as well as giving theproteoglycan it’s overall negative charge.

4. The Golgi is also capable of phosphorylating molecules. To do so ittransports ATP into the lumen. The Golgi itself contains resident kinases,such as casein kinases. One molecule that is phosphorylated in the Golgi isApolipoprotein, which forms a molecule known as VLDL that is a constitute ofblood serum. It is thought that the phosphorylation of these molecules isimportant to help aid in their sorting of secretion into the blood serum.

5. The Golgi also has a putative role in apoptosis, with several Bcl-2family members localised there, as well as to the mitochondria. In addition anewly characterised anti-apoptotic protein, GAAP (Golgi anti-apoptotic protein),which almost exclusively resides in the Golgi, protects cells from apoptosis byan as-yet undefined mechanism (Gubser et al., 2007).

VESICULAR TRANSPORTVesicles which leave the rough endoplasmic reticulum are transported to

the cis face of the Golgi apparatus, where they fuse with the Golgi membraneand empty their contents into the lumen. Once inside they are modified,sorted, and shipped towards their final destination. As such, the Golgiapparatus tends to be more prominent and numerous in cells synthesisingand secreting many substances: plasma B cells, the antibody-secreting cellsof the immune system, have prominent Golgi complexes.

Those proteins destined for areas of the cell other than either theendoplasmic reticulum or Golgi apparatus are moved towards the trans face,to a complex network of membranes and associated vesicles known as thetrans-Golgi network (TGN). This area of the Golgi is the point at which proteins

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are sorted and shipped to their intended destinations by their placement intoone of at least three different types of vesicles, depending upon the molecularmarker they carry.

Fig. 10. : Inner view of Golgi Apparatus

TRANSPORT MECHANISMThe transport mechanism which proteins use to progress through the

Golgi apparatus is not yet clear; however a number of hypotheses currentlyexist. Until recently, the vesicular transport mechanism was favoured butnow more evidence is coming to light to support cisternal maturation. Thetwo proposed models may actually work in conjunction with each other, ratherthan being mutually exclusive. This is sometimes referred to as the combinedmodel.

Cisternal maturation model: The cisternae of the Golgi apparatus moveby being built at the cis face and destroyed at the trans face. Vesicles from theendoplasmic reticulum fuse with each other to form a cisterna at the cis face,

* consequently this cisterna would appear to move through the Golgistack when a new cisterna is formed at the cis face. This model issupported by the fact that structures larger than the transportvesicles, such as collagen rods, were observed microscopically toprogress through the Golgi apparatus. This was initially a popularhypothesis, but lost favour in the 1980s. Recently it has made acomeback, as laboratories at the University of Chicago and theUniversity of Tokyo have been able to use new technology to directlyobserve Golgi compartments maturing. Additional evidence comes fromthe fact that COP1 vesicles move in the retrograde direction,.transporting ER proteins back to where they belong by recognizing asignal peptide.

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* Vesicular transport model: Vesicular transport views the Golgi as avery stable organelle, divided into compartments is the cis to transdirection. Membrane bound carriers transported material betweenthe ER and Golgi and the different compartments of the Golgi.Experimental evidence inlcudes the abundance of small vesicles(known technically as shuttle vesicles) in proximity to the Golgiapparatus. Directionality is achieved by packaging proteins into eitherforward-moving or backward-moving (retrograde) transport vesicles,or alternatively this directionality may not be necessary as theconstant input of proteins from the endoplasmic reticulum on the cisface of the Golgi would ensure flow. Irrespectively, it is likely that thetransport vesicles are connected to a membrane via actin filamentsto ensure that they fuse with the correct compartment.

2.5 RIBOSOMESRibosomes were first observed in the mid-1950s by Romanian cell biologist

George Palade in the electron microscope as dense particles or granules forwhich he was awarded the Nobel Prize. The term ribosome was proposed byscientist Richard B. Roberts in 1958. A ribosome is a small, dense, functionalstructure found in all known cells that assembles proteins. They are about20nm in diameter and are composed of 65% ribosomal RNA and 35% ribosomalproteins (known as a Ribonucleoprotein or RNP). It translates messenger RNA(mRNA) to build a polypeptide chain (e.g., a protein) using amino acids deliveredby Transfer RNA (tRNA). It can be thought of as a giant enzyme but, althoughit contains proteins, its active site is made of RNA, so ribosomes are nowclassified as “ribozymes”.

Ribosomes build proteins from the genetic instructions held within amessenger RNA. Free ribosomes are suspended in the cytosol (the semi-fluidportion of the cytoplasm) or bound to the rough endoplasmic reticulum, or tothe nuclear envelope. Since ribosomes are ribozymes, it is thought that theymight be remnants of the RNA world. While catalysis of the peptide bondinvolves the C2' hydroxyl of tRNA’s P-site adenosine in a sort of proton shuttlemechanism, the full function (ie, translocation) of the ribosome is reliant onchanges in protein conformations.

Fig. 11. : Structure of Ribosome

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Ribosomes consist of two subunits that fit together and work as one totranslate the mRNA into a polypeptide chain during protein synthesis.Prokaryotic subunits consist of one or two and eukaryotic of one or three verylarge RNA molecules (known as ribosomal RNA or rRNA) and multiple smallerprotein molecules. Prokaryotes have 70S ribosomes, each consisting of a small(30S) and a large (50S) subunit. Their large subunit is composed of a 5S RNAsubunit (consisting of 120 nucleotides), a 23S RNA subunit (2900 nucleotides)and 34 proteins. The 30S subunit has a 1540 nucleotide RNA subunit boundto 21 proteins. Eukaryotes have 80S ribosomes, each consisting of a small(40S) and large (60S) subunit. Their large subunit is composed of a 5S RNA(120 nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit (160nucleotides) and ~49 proteins. The 40S subunit has a 1900 nucleotide (18S)RNA and ~33 proteins. Crystallographic work has shown that there are noribosomal proteins close to the reaction site for polypeptide synthesis. Thissuggests that the protein components of ribosomes act as a scaffold that mayenhance the ability of rRNA to synthesize protein rather than directlyparticipating in catalysis.Free ribosomes: Free ribosomes are “free” to moveabout anywhere in the cytoplasm (within the cell membrane). Proteins madeby free ribosomes are used within the cell. Proteins containing disulfide bondsusing cysteine amino acids cannot be produced outside of the lumen of theendoplasmic reticulum.

Membrane-bound ribosomes: When certain proteins are synthesized bya ribosome they can become “membrane-bound”. The newly producedpolypeptide chains are inserted directly into the endoplasmic reticulum bythe ribosome and are then transported to their destinations. Bound ribosomesusually produce proteins that are used within the cell membrane or areexpelled from the cell via exocytosis.

FUNCTION OF RIBOSOMES1. Ribosomes are the workhorses of protein biosynthesis, the process of

translating messenger RNA (mRNA) into protein. The mRNA comprisesa series of codons that dictate to the ribosome the sequence of theamino acids needed to make the protein. Using the mRNA as atemplate, the ribosome traverses each codon of the mRNA, pairing itwith the appropriate amino acid. This is done using molecules oftransfer RNA (tRNA) containing a complementary anticodon on oneend and the appropriate amino acid on the other.

2. Protein synthesis begins at a start codon near the 5' end of the mRNA.The small ribosomal subunit, typically bound to a tRNA containingthe amino acid methionine, binds to an AUG codon on the mRNA andrecruits the large ribosomal subunit. The large ribosomal subunitcontains three tRNA binding sites, designated A, P, and E. The A sitebinds an aminoacyl-tRNA (a tRNA bound to an amino acid); the P sitebinds a peptidyl-tRNA (a tRNA bound to the peptide being synthesized);and the E site binds a free tRNA before it exits the ribosome.

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Both ribosomal subunits (small and large) assemble at the start codon(towards the 5' end of the mRNA). The ribosome uses tRNA which matchesthe current codon (triplet) on the mRNA to append an amino acid to thepolypeptide chain. This is done for each triplet on the mRNA, while the ribosomemoves towards the 3' end of the mRNA. Usually in bacterial cells, severalribosomes are working parallel on a single mRNA, forming what we call apolyribosome or polysome

2.6 LYSOSOMESLysosomes are organelles that contain digestive enzymes (acid

hydrolases). They digest excess or worn out organelles, food particles, andengulfed viruses or bacteria. The membrane surrounding a lysosome preventsthe digestive enzymes inside from destroying the cell. Lysosomes fuse withvacuoles and dispense their enzymes into the vacuoles, digesting theircontents. They are built in the Golgi apparatus. The name lysosome derivesfrom the Greek words lysis, which means dissolution or destruction, andsoma, which means body. They are frequently nicknamed “suicide-bags” or“suicide-sacs” by cell biologists due to their role in autolysis. Lysosomes werediscovered by the Belgian cytologist Christian de Duve in 1949.

ACIDIC ENVIRONMENTAt pH 4.8, the interior of the lysosomes is more acidic than the cytosol

(pH 7.2). The lysosome single membrane stabilizes the low pH by pumping inprotons (H+) from the cytosol via proton pumps and chloride ion channels. Themembrane also protects the cytosol, and therefore the rest of the cell, fromthe degradative enzymes within the lysosome. For this reason, should alysosome’s acid hydrolases leak into the cytosol, their potential to damagethe cell will be reduced, because they will not be at their optimum pH. Thehydrolytic enzymes in lysosomes are produced in the endoplasmic reticulum

Fig. 13. : Lysosome

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and transported and processed through the Golgi apparatus. The Golgiapparatus produces lysosomes by budding. Each acid hydrolase is then targetedto a lysosome by phosphorylation. The lysosome itself is likely to be safe fromenzymatic action due to having proteins in the inner membrane which has athree-dimensional molecular structure that protects vulnerable bonds fromenzymatic attack.

Some important enzymes in lysosomes are:* Lipase, which digests lipids,* Carbohydrases, which digest carbohydrates (e.g., sugars),* Proteases, which digest proteins,* Nucleases, which digest nucleic acids.* Phosphatases, which digest phosphoric acid monoesters

Lysosomal enzymes are synthesized in the cytosol and the endoplasmicreticulum, where they receive a mannose-6-phosphate tag that targets themfor the lysosome. Aberrant lysosomal targeting causes inclusion-cell disease,whereby enzymes do not properly reach the lysosome, resulting inaccumulation of waste within these organelles.

FUNCTIONSThe lysosomes are used for the digestion of macromolecules from

phagocytosis (ingestion of other dying cells or larger extracellular material),endocytosis (where receptor proteins are recycled from the cell surface), andautophagy (where old or unneeded organelles or proteins, or microbes whichhave invaded the cytoplasm are delivered to the lysosome). Autophagy mayalso lead to autophagic cell death, a form of programmed self-destruction, orautolysis, of the cell, which means that the cell is digesting itself.

Other functions include digesting foreign bacteria (or other forms of waste)that invade a cell and helping repair damage to the plasma membrane byserving as a membrane patch, sealing the wound. Lysosomes also do much ofthe cellular digestion required to digest tails of tadpoles and to remove theweb from the fingers of a 3-6 month old fetus. This process of programmedcell death is called apoptosis.

2.7 NUCLEUSIn cell biology, the nucleus (pl. nuclei; from Latin nucleus or nuculeus,

kernel) is a membrane-enclosed organelle found in most eukaryotic cells. Itcontains most of the cell’s genetic material, organized as multiple long linearDNA molecules in complex with a large variety of proteins, such as histones,to form chromosomes. The genes within these chromosomes make up thecell’s nuclear genome. The function of the nucleus is to maintain the integrityof these genes and to control the activities of the cell by regulating geneexpression.

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Fig. 13. : Nuclear

The main structural elements of the nucleus are the nuclear envelope, adouble membrane that encloses the entire organelle and keeps its contentsseparated from the cellular cytoplasm, and the nuclear lamina, a meshworkwithin the nucleus that adds mechanical support much like the cytoskeletonsupports the cell as a whole. Because the nuclear membrane is impermeableto most molecules, nuclear pores are required to allow movement of moleculesacross the envelope. These pores cross both membranes of the envelope,providing a channel that allows free movement of small molecules and ions.The movement of larger molecules such as proteins is carefully controlled,and requires active transport facilitated by carrier proteins. Nuclear transportis of paramount importance to cell function, as movement through the poresis required for both gene expression and chromosomal maintenance.

Although the interior of the nucleus does not contain any membrane-delineated bodies, its contents are not uniform, and a number of subnuclearbodies exist, made up of unique proteins, RNA molecules, and DNAconglomerates. The best known of these is the nucleolus, which is mainlyinvolved in assembly of ribosomes. After being produced in the nucleolus,ribosomes are exported to the cytoplasm where they translate mRNA.

2.8 NUCLEOLUSThe nucleolus is a discrete densely-stained structure found in the nucleus.

It is not surrounded by a membrane, and is sometimes called a suborganelle.It forms around tandem repeats of rDNA, DNA coding for ribosomal RNA (rRNA).These regions are called nucleolar organizer regions (NOR). The main rolesof the nucleolus are to synthesize rRNA and assemble ribosomes. The structuralcohesion of the nucleolus depends on its activity, as ribosomal assembly inthe nucleolus results in the transient association of nucleolar components,facilitating further ribosomal assembly, and hence further association. Thismodel is supported by observations that inactivation of rDNA results inintermingling of nucleolar structures.

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Fig. 14. : Nucleolus

The first step in ribosomal assembly is transcription of the rDNA, by aprotein called RNA polymerase I, forming a large pre-rRNA precursor. This iscleaved into the subunits 5.8S, 18S, and 28S rRNA. The transcription, post-transcriptional processing, and assembly of rRNA occurs in the nucleolus,aided by small nucleolar RNA (snoRNA) molecules, some of which are derivedfrom spliced introns from messenger RNAs encoding genes related to ribosomalfunction. The assembled ribosomal subunits are the largest structures passedthrough the nuclear pores.

When observed under the electron microscope, the nucleolus can be seento consist of three distinguishable regions: the innermost fibrillar centers (FCs),surrounded by the dense fibrillar component (DFC), which in turn is borderedby the granular component (GC). Transcription of the rDNA occurs either in theFC or at the FC-DFC boundary, and therefore when rDNA transcription in thecell is increased more FCs are detected. Most of the cleavage and modificationof rRNAs occurs in the DFC, while the latter steps involving protein assemblyonto the ribosomal subunits occurs in the GC.

2.9 PEROXISOMESPeroxisomes are ubiquitous organelles in eukaryotes that participate in

the metabolism of fatty acids and other metabolites. Peroxisomes have enzymesthat rid the cell of toxic peroxides. They have a single lipid bilayer membranethat separates their contents from the cytosol (the internal fluid of the cell)and contain membrane proteins critical for various functions, such as importingproteins into the organelles and aiding in proliferation. Like lysosomes,peroxisomes are part of the secretory pathway of a cell, but they are muchmore dynamic and can replicate by enlarging and then dividing. Peroxisomeswere identified as cellular organelles by the Belgian cytologist Christian deDuve in 1965 after they had been first described in a Swedish Ph.D. thesis adecade earlier.

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Fig. 15. : Anatomy of the Peroxisome

FUNCTION OF PEROXISOMESPeroxisomes contain oxidative enzymes, such as catalase, D-amino acid

oxidase and uric acid oxidase. Certain enzymes within the peroxisome, byusing molecular oxygen, remove hydrogen atoms from specific organicsubstrates (labeled as R), in an oxidative reaction, producing hydrogen peroxide(H2O2, itself toxic):

. . . . . (1)

Catalase, another enzyme in the peroxisome, in turn uses this H2O2 tooxidize other substrates, including phenols, formic acid, formaldehyde andalcohol, by means of the peroxidation reaction:

, . . . (2)

thus eliminating the poisonous hydrogen peroxide in the process.

This reaction is important in liver and kidney cells where the peroxisomesdetoxifiy various toxic substances that enter the blood. About 25% of theethanol we drink is oxidized to acetaldehyde in this way. In addition, whenexcess H2O2 accumulates in the cell, catalase converts it to H2O through thisreaction:

. . . (3)

A major function of the peroxisome is the breakdown of fatty acidmolecules, in a process called beta-oxidation. In this process, the fatty acidsare broken down two carbons at a time, converted to Acetyl-CoA, which isthen transported back to the cytosol for further use. In animal cells, beta-oxidation can also occur in the mitochondria. In yeast and plant cells thisprocess is exclusive for the peroxisome.The first reactions in the formation ofplasmalogen in animal cells also occurs in peroxisomes. Plasmalogen is themost abundant phospholipid in myelin. Deficiency of plasmalogens causesprofound abnormalities in the myelination of nerve cells, which is one of the

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reasons that many peroxisomal disorders lead to neurologicaldisease.Peroxisomes also play a role in the production of bile acids.

PROTEIN IMPORTProteins are selectively imported into peroxisomes. Since the organelles

contain no DNA or ribosomes and thus have no means of producing proteins,all of their proteins must be imported across the membrane. It is believedthat proteins do not transit through the endoplasmic reticulum to get to theperoxisome.

A specific protein signal (PTS or peroxisomal targeting signal) of threeamino acids at the C-terminus of many peroxisomal proteins signals themembrane of the peroxisome to import them into the organelle. Otherperoxisomal proteins contain a signal at the N-terminus. There are at least 32known peroxisomal proteins, called peroxins, which participate in the processof importing proteins by means of ATP hydrolysis. Proteins do not have tounfold to be imported into the peroxisome. The protein receptors, the peroxinsPex5 and Pex7, accompany their cargoes (containing a PTS1 or a PTS2,respectively) all the way into the peroxisome where they release the cargoand then return to the cytosol - a step named “recycling”. Overall, the importcycle is referred to as the “extended shuttle mechanism”. Evidence nowindicates that ATP hydrolysis is required for the recycling of receptors to thecytosol. Also, ubiquitination appears to be crucial for the export of PEX5 fromthe peroxisome, to the cytosol. Little is known about the import of PEX7,although it has helper proteins that have been shown to be ubiquitinated.

Deficiencies: Peroxisomal disorders are a class of conditions which leadto disorders of lipid metabolism. One well known example is Zellwegersyndrome.One of these is called a tight junction or “occluding junction” (zonulaoccludens). This is shown as the top junction in the above drawing. At thissite, membrane glycoproteins and associated “glue” bind the cells togetherlike double-sided “strapping tape”.

2.10 LET US SUM UP1. Mitochondria are the cells’ power house of the cells2. the high energy compound ATP is produced in the mitochondria3. Glucose is breakdown by two pathways in mitochondria. Anaerobic

metabolism and aerobic metabolism.4. Kreb cycle and oxidative phosphorylation takes place in mitochondria5. The chloroplast consists of an inner and an outer phospholipid

membrane6. Main function is photosynthesis.7. Endoplasmic reticulum is responsible for several specialized functions

like Protein translation, folding.8. There are three varieties of endoplasmic reticulum they are rough

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endoplasmic reticulum, smooth endoplasmic reticulum, andsarcoplasmic reticulum.

9. The surface of the rough endoplasmic reticulum is studded withprotein-manufacturing ribosomes giving it a “rough” appearance

10. Prokaryotes have 70S ribosomes, each consisting of a small (30S) anda large (50S) subunit.

11. Lysosomes are organelles that contain digestive enzymes (acidhydrolases) to digest excess or worn out organelles, food particles,and engulfed viruses or bacteria.

12. The nucleolus consists of three distinguishable regions: the innermostfibrillar centers, surrounded by the dense fibrillar component, whichin turn is bordered by the granular component.

13. Peroxisomes are ubiquitous organelles in eukaryotes that participate in themetabolism of fatty acids and other metabolites.

14. Peroxisomes contain oxidative enzymes, such as catalase, D-amino acid oxidaseand uric acid oxidase.

2.11 POINTS FOR DISCUSSION* “Exploring cell organelles is as important as knowing the cell itself” –

Substantiate.

2.12 SELF-CHECK EXERCISEDiscuss the structure of mitochodria and its importance as a power houseNote: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer.............................................................................................................................................................................................................................................................................................................................................................................................................................................................

2.13 LEESSION-END ACTIVITIES1) What are the different types of endoplasmic reticulum?2) Write about the main functions of endoplasmic reticulum3) Define the terms Exocytotic vesicles, Secretory vesicles, Lysosomal

vesicles4) How the molecules are transported across the golgi complex?5) What is zone of exclusion?6) What are the subunits of ribosome?7) Explain the role of ribosomes in protein synthesis.8) What are the different enzymes present in Lysosomes?9) Give short notes on the functions of Lysosome?

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10) What is nucleolus?11) Give short notes on the structure of nucleolus.






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LESSON - 3

CELL MEMBRANE STRUCTURE AND TRANSPORT PROTEINS

CONTENTS


3.1. Membrane Structure

3.2 Let us sum up


3.4 Self check exercise


3.6 References

3.0 AIMS AND OBJECTIVESTo know about cell membrane structure and transport proteins.

3.1. MEMBRANE STRUCTUREThe cell is highly organized with many functional units or organelles.

Most of these units are limited by one or more membranes. To perform thefunction of the organelle, the membrane is specialized in that it containsspecific proteins and lipid components that enable it to perform its uniqueroles for that cell or organelle. In essence membranes are essential for theintegrity and function of the cell. Membrane components may:

a) be protectiveb) regulate transport in and out of cell or subcellular domainc) allow selective receptivity and signal transduction by providing

transmembrane receptors that bind signaling moleculesd) allow cell recognitione ) Provide anchoring sites for cytoskeletal filaments or components of

the extracellular matrix. This allows the cell to maintain its shapeand perhaps move to distant sites.

f) help compartmentalize subcellular domains or microdomainsg) Provide a stable site for the binding and catalysis of enzymes.h) regulate the fusion of the membrane with other membranes in the

cell via specialized junctions )i) Provide a passageway across the membrane for certain molecules,

such as in gap junctions.

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j) allow directed cell or organelle motility

Membrane theories: In the early 1930’s-40’s, Danielli and Davson studiedtriglyceride lipid bilayers over a water surface. They found them to arrangethemselves with the polar heads facing outward. However, they always formeddroplets (oil in water) and the surface tension was much higher than that ofcells. However, if proteins were added the surface tension was reduced andthe membranes flattened out.

Fig. 16 : Cell Membrane

In the 1950’s Robertson noted the structure of membranes seen in theabove electron micrographs. He saw no spaces for pores in the electronmicrographs. He hypothesized that the railroad track appearance came fromthe binding of osmium tetroxide to proteins and polar groups of lipids.

Fig. 17: Inner View of Cell Membrane

FLUID-MOSAIC MODEL:Biological membranes are sheet-like structures composed mainly of lipids

and proteins. All biological membranes have a similar general structure.Membrane lipids are organized in a bilayer (two sheets of lipids making up asingle membrane) that is approximately 60 to 100 Å thick. The proteins, onthe other hand, are scattered throughout the bilayer and perform mostmembrane functions. Membranes are two-dimensional fluids: both lipids andproteins are constantly in motion. The fluid-mosaic model encompasses ourcurrent understanding of membrane structure. It describes both the “mosaic”arrangement of proteins embedded throughout the lipid bilayer as well as the“fluid” movement of lipids and proteins alike.

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Fig. 19 :Reaction of Cell Membranes

Membrane Phospholipids: One of the principal types of lipid in themembrane include the phospholipids . These have a polar head group and twohydrocarbon tails. An example of a phospholipid is shown in this figure (right).The top region beginning with the NH3 is the polar group. It is connected byglycerol to two fatty acid tails. One of the tails is a straight chain fatty acid(saturated). The other has a link in the tail because of a cis double bond(unsaturated).The lipid bilayer gives the membranes its fluid characteristics.The following figure shows the effect of temperature on the packing of thehydrocarbons. Note that a low temperatures, the bilayer is in a gel state andtightly packed. At higher (body) temperatures, the bilayer actually “melts’and the interior is fluid allowing the lipid molecules to move around, rotate,exchange places.

Fig. 20 : Reaction of Cell Membranes

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Membrane Cholesterol: Another type of lipid in the membrane ischolesterol. The amount of cholesterol may vary with the type of membrane.Plasma membranes have nearly one cholesterol per phospholipid molecule.Other membranes (like those around bacteria) have no cholesterol. Thecholesterol molecule inserts itself in the membrane with the same orientationas the phospholipid molecules. The figures show phospholipid molecules witha cholesterol molecule inbetween. Note that the polar head of the cholesterolis aligned with the polar head of the phospholipids. Cholesterol moleculeshave several functions in the membrane: a) They immobilize the first fewhydrocarbon groups of the phospholipid molecules. This makes the lipid bilayerless deformable and decreases its permeability to small water-solublemolecules. Without cholesterol (such as in a bacterium) a cell would need acell wall b) Cholesterol prevents crystallization of hydrocarbons and phaseshifts in the membrane.

Membrane Glycolipids: Glycolipids are also a constituent of membraneswhich projecting into the extracellular space and hereby serving as protective,insulators, and sites of receptor binding. Among the molecules bound byglycososphingolipids include cell poisons such as cholera and tetanustoxins.Formation of “Microdomains”: Sphingolipids and cholesterol worktogether to help cluster proteins in a region called a “microdomain”. Theyfunction as “rafts” or platforms for the attachment of proteins as membranesare moved around the cell and also during signal transduction.

Membrane Proteins: Transmembrane proteins are amphipathic, in thatthey have hydrophobic and hydrophilic regions that are oriented in the sameregions in the lipid bilayer. Another name for them is “integral proteins”.Other types of proteins may be linked only at the cytoplasmic surface (byattachment to a fatty acid chain), or at the external cell surface, attached bya oligosaccharide. Or, these non-transmembrane proteins may be bound toother membrane proteins. Collectively these are called “peripheral membraneproteins”. We will be studying specific membrane proteins in later lectures(ion channels, proteins in endoplasmic reticulum, etc). Therefore, thispresentation will not spend much time on them. Proteins inserted oncethrough the membrane are called “single-pass transmembrane proteins.” Thosethat pass through several times are called “multipass transmembraneproteins” and form loops outside the membrane

Fig. 20 : Inner View of Cell Membrane

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3.2 LET US SUM UP1. Membrane is specialized in that it contains specific proteins and lipid

components to perform the function of the organelle2. various membrane theories have been proposed3. Danielli and Davson, Robertson model, and Fluid mosaic model have

been proposed4. Membrane transport like facilitated diffusion, active transport,

channels and pores, active transporters have been discussed.5. Phagocytosis, pinocytosis, endocytosis are also discussed.

3.3 POINTS FOR DISCUSSIONDo an analysis of the structure of the cell membrane and highlight the

interesting teachers of it.

3.4 CHECK YOUR PROGRESSExplain the models proposed for membrane strcuture?Note: a) Please don’t proceed till you attempt the above question.


3.5 LESSON-END ACTIVITIES1) Which component(s) of membranes give it its fluid characteristics?2) What feature in a membrane helps to prevent freezing? Be specific.3) Which part of a membrane helps it keep its shape (prevents

deformation)?4) How are proteins arranged in a membrane? What is the difference

between a transmembrane protein and a peripheral membraneprotein?

5) What is a microdomain, and how is it formed?6) If one type of membrane contains 76% proteins and another type

contains only 18% proteins, what might you conclude about functionaldifferences? For example, see Membrane Architecture

7) What experiments might you conduct to prove that proteins moved inthe plane of the membrane?

8) How do membranes support the polarity of a cell?9) How would you detect receptors in the plasma membrane of a cell?10) In a freeze-fracture/freeze etch specimen, what are the bumps seen

in the plane of the membrane?

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11) How would you distinguish tight, or occluding junction between twocells, both structurally and functionally.

12) What experiments would you use to prove cells were communicatingvia gap junctions? Do you know how gap junctions are formed?

13) What does the presence of microvilli signify?14) What experimental approach could you use to show that a protein is

inserted in the membrane?

3.6 REFERENCES

1. Lehinger, A.L. 1984, Principles of Biochemistry, CBS Publishers anddistributors, New Delhi, India.

2. Horton, Moran, Ochs, Rawn, Scrimgeour Principles of Biochemistry,Prentice Hall Publishers.

3. Shanmughavel, P. 2005, Principles of Bioinformatics, PointerPublishers, Jaipur, India.

4. David, E. Sadava Cell Biology: Organelle structure and Fucntion Jones& Bartlett Publishers.

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LESSON - 4

MECHANISMS OF TRANSPORT

CONTENTS


4.1 Facilitated diffusion

4.2 Active transport

4.3 Let us sum up




4.7 References

4.0 AIMS AND OBJECTIVESTo know about mechanisms of transports and its branches of transports.

4.1 FACILITATED DIFFUSIONA facilitated diffusion protein speeds the movement of a chemical through

a membrane in the absence of energy input; therefore, the transportedchemical can only move down a concentration gradient. This can beaccomplished by the formation of a high-specificity pore or channel that spansthe membrane.

4.2 ACTIVE TRANSPORT:Transport proteins are also used in active transport, which by definition

does require an energy input. Chemiosmotic transport utilizes electrochemicalgradients to drive transport. As the creation and maintenance of chemiosmoticgradients require energy input from the cell, this is a form of active transport.Prokaryotes typically use hydrogen ions as the driving force for chemiosmotictransport, while eukaryotes typically use sodium ions. A symporter/coportertransports a chemical in the same direction as the electrochemical gradient,while an antiporter moves the target chemical in a direction opposite to thegradient.The uniporter is also often included as a category of chemiosmotictransporter, although a uniporter can also be considered as a facilitateddiffusion protein on the basis of function.

Binding dependent active transport: Binding dependent active transport

36

also moves the targeted chemical against a concentration gradient, but usesstored chemical energy, typically in the form of adenosine triphosphate, topower the transport. Generally speaking, a binding dependent transport systemconsists of a membrane spanning component with a high degree of specifity.The membrane spanning component changes configuration with the aid ofchemical energy input, thus translocating the chemical from one side of themembrane to the other.

CHANNELS/PORES

Voltage-gated ion channel like, including potassium channels KcsAand KvAP, and inward-rectifier potassium ion channel Kirbac

Large-conductance mechanosensitive channel, MscL

Small-conductance mechanosensitive ion channel (MscS)

CorA metal ion transporters

Ligand-gated ion channel of neurotransmitter receptors (acetylcholinereceptor) Aquaporins

Chloride channels

Outer membrane auxiliary proteins (polysaccharide transporter)

Electrochemical Potential-driven transporters

Mitochondrial carrier proteins

Major Facilitator Superfamily (Glycerol-3-hosphate transporter,Lactose permease, and Multidrug transporter EmrD)

Resistance-nodulation-cell division (multidrug efflux transporter AcrB)

Dicarboxylate/amino acid:cation symporter (proton glutamatesymporter)

Monovalent cation/proton antiporter (Sodium/proton antiporter 1 NhaA)

Neurotransmitter sodium symporter

Ammonia transporters

Drug/Metabolite Transporter (small multidrug resistance transporterEmrE)

Primary Active Transporters

Light absorption-driven transporters:

Bacteriorhodopsin-like proteins including rhodopsin (see also opsin)

Bacterial photosynthetic reaction centres and photosystems I and II

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Light harvesting complexes from bacteria and chloroplasts

Oxidoreduction-driven transporters

Transmembrane cytochrome b-like proteins: coenzyme Q - cytochromec reductase (cytochrome bc1 ); cytochrome b6f complex; formatedehydrogenase, respiratory nitrate reductase; succinate - coenzymeQ reductase (fumarate reductase); and succinate dehydrogenase.

Cytochrome c oxidases from bacteria and mitochondria

Electrochemical potential-driven transporters

Proton or sodium translocating F-type and V-type ATPases

P-P-bond hydrolysis-driven transporters

P-type calcium ATPase (five different conformations)

Calcium ATPase regulators phospholamban and sarcolipin

ABC transporters: BtuCD, multidrug transporter, and molybdateuptake transporter

General secretory pathway (Sec) translocon (preprotein translocaseSecY)

ACCESSORY FACTORS INVOLVED IN TRANSPORTEndocytosis is a process whereby cells absorb material (molecules such

as proteins) from the outside by engulfing it with their cell membrane. It isused by all cells of the body because most substances important to them arelarge polar molecules, and thus cannot pass through the hydrophobic plasmamembrane. The function of endocytosis is the opposite of exocytosis.

Fig. 21: Endocytosis

The absorption of material from the outside environment of the cell iscommonly divided into two processes: phagocytosis and pinocytosis.

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Phagocytosis (literally, cell-eating) is the process by which cells ingestlarge objects, such as cells which have undergone apoptosis, bacteria, orviruses. The membrane folds around the object, and the object is sealed offinto a large vacuole known as a phagosome.

Fig. 22 : Phagocytosis

Pinocytosis (literally, cell-drinking) is a synonym for endocytosis. Thisprocess is concerned with the uptake of solutes and single molecules such asproteins.

Fig. 23 : Pinocytosis

Receptor-mediated endocytosis is a more specific active event where thecytoplasm membrane folds inward to form coated pits. These inward buddingvesicles bud to form cytoplasmic vesicles.

4.3 LET US SUM UPMechanism of transport to produce facilitates diffusion.Various transports are activated in this lesson.Channels/pores are to help them transporters.

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4.4 POINTS FOR DISCUSSIONComment on the various transport mechanisms in a cell.

4.5 CHECK YOUR PROGRESSExplain the models proposed for mechanism of transport?Note: a) Please don’t proceed till you attempt the above question.


4.6 LESSON-END ACTIVITIES1. What is meant by membrane transport protein?2. Define Channels/pores.3. What are the electrochemical potential-driven transporters?4. Which are primary active transporters?

4.7 REFERENCES





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41

UNIT - II

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43

LESSON - 5

CARBOHYDRATES

CONTENTS


5.1 Introduction to carbohydrates

5.2 Types of carbohydrates

Monosaccharides

Disaccharides

Polysaccharides

5.4 Importance of Carbohydrates

5.5 Let us sum up




5.9 References

5.0 AIMS AND OBJECTIVESTo know about the significance of carbohydrates and its types.

5.1 INTRODUCTION TO CARBOHYDRATESThe monosaccharides traditionally referred to a class of

polyhydroxylcarbonyl compounds that had the empirical formula CH2O. Thisdefinition has been expanded to include compounds that may contain certainoxidized, reduced, or heteroatom-substituted groups. Carboydrates are builtfrom these monosaccharide units. A sugar may refer to a monosaccharide orto small compounds with more than one monosaccharide. A polysaccharidecontains many monosaccharide units.The suffix -ose indicates a sugar.Hexoses are six-carbon sugars and pentoses are five-carbon sugars. Manymonosaccharides, such as glucose, may be in equilibrium between their acyclicand cyclic form. From their acyclic form the terms aldose and ketose arederived. An aldose will contain an aldeyde and a ketose contains a ketone.The monosaccharide may also be referred to its cyclic form. If the ring hasfive carbons and one oxygen, it is a pyranose. If it has four carbons and one

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oxygen, it is a furanose.Carbos = Sugars: C, H, and O in 1:2:1 ratio (roughlyCH2O).

5.2 TYPES OF CARBOHYDRATES:Monosaccharides: simple sugars (the building block of all larger sugars)

C6H12O6. Because of their six carbon atoms, each is a hexose. Examples ofMonosaccharides: Glucose - Form of simple sugar used by all cells. Fromgrapes & honey, Fructose - Fruit sugar, Galactose, a sugar in milk (andyogurt).

Fig. 24 : Monosaccharide

Although all three share the same molecular formula (C6H12O6), thearrangement of atoms differs in each case. Substances such as these three,which have identical molecular formulas but different structural formulas,are known as structural isomers

* Disaccharides: double sugars, formed by dehydration synthesis(removal of water as the 2 monosaccharides bond), Maltose = glucose+ glucose, Sucrose = glucose + fructose, Lactose = glucose + galactose.

* Polysaccharides: starches, chains of sugars ,formed by dehydrationsynthesis, examples: Amylose, Pectins, Glycogen, Cellulose,undigestible by most organisms.

Fig. 25 : Polysaccharides

5.3 IMPORTANCE OF CARBOHYDRATESGlucose - key metabolic fuel (energy source) of all cells. Animal Starch

(Glycogen)- long term energy storage for animal cells (stores the glucose

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molecules in a form not easily used).Plant Starch (Amylose) - long term energystorage for plant cells. Cellulose - Structural polysaccharide of cell walls,Chitin - Structural polysaccharide of exoskeletons of insects and crustaceans.

Ribose is the 5 carbon sugar found in RNA (ribonucleic acid).

5.4 LET US SUM UPCarboydrates are built from these monosaccharide units. There are three

different types carbohydrates such as Monosaccharides, Disaccharides,Polysaccharides. They play important roles in organisms acting as energysources for different activities.

5.5 POINTS FOR DISCUSSION“Carbohydrates are essential metabolic fuel of all cells” – Express your

views on this.

5.6 CHECK YOUR PROGRESSGive an account on polysaccharides?Note: a) Please don’t proceed till you attempt the above question.


5.7 LESSON-END ACTIVITIES1) Notes on introduction to carbohydrate.2) Explain the types of Carbohydrates.3) What is the importance of Carbohydrates in living systems?


distributors, New Delhi, India.

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LESSON - 6

PROTEINS

CONTENTS6.0 Aims and Objectives

6.1 Introduction

6.2 Amino Acids

6.3 Different level of organization

Primary Structure

Secondary Structure

6.4 Let us sum up




6.8 References

6.0 AIMS AND OBJECTIVESTo learn the importance of proteins and amino acids and their different

levels of organization.

6.1 INTRODUCTIONProteins are large organic compounds made of amino acids arranged in a

linear chain and joined together by peptide bonds between the carboxyl andamino groups of adjacent amino acid residues. The sequence of amino acidsin a protein is defined by a gene and encoded in the genetic code. Althoughthis genetic code specifies 20 “standard” amino acids, the residues in a proteinare often chemically altered in post-translational modification: either beforethe protein can function in the cell, or as part of control mechanisms. Proteinscan also work together to achieve a particular function, and they oftenassociate to form stable complexes. Like other biological macromolecules suchas polysaccharides and nucleic acids, proteins are essential parts of organismsand participate in every process within cells. Many proteins are enzymes thatcatalyze biochemical reactions, and are vital to metabolism. Proteins alsohave structural or mechanical functions, such as actin and myosin in muscle,and the proteins in the cytoskeleton, which forms a system of scaffolding thatmaintains cell shape. Other proteins are important in cell signaling, immune

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responses, cell adhesion, and the cell cycle. Protein is also a necessary partof animals’ diets, since they cannot synthesise all the amino acids and mustobtain essential amino acids from food. Through the process of digestion,animals break down ingested protein into free amino acids that can be usedfor protein synthesis

6.2 AMINO ACIDSAn amino acid is by definition an organic compound containing an amine

group and a carboxylic acid group in the same molecule. While there aremany forms of amino acids, all of the important amino acids found in livingorganisms are alpha-amino acids. Alpha amino acids have the -COOH and -NH2 groups both attached to the same carbon atom. The simplest amino acid,which is the molecule glycine, H2NCH2COOH, contains no asymmetric carbonatoms (tetrahedral carbon atoms with four different groups attached). All ofthe other amino acids do contain such a carbon atom and are thereforeoptically active. The general structure of the alpha-amino acids is R-CHNH2(alpha)-COOH, and optical activity for the alpha-amino acids morecomplex than glycine is found at the alpha carbon. Amino acids have both anacidic group, in the carboxylic acid, and a basic group, in the amine. Underphysiological aqueous conditions a proton transfer from the acid to the baseoccurs, forming a dipolar ion or zwitterion, because the carboxylic acid is amuch stronger acid than is the ammonium ion. The actual structure of glycinein solution, for example, is +H3NCH2COO- at pH 7 rather thanH2NCH2COOH.At very low pH the acid group can be protonated and at veryhigh pH the ammonium group can be deprotonated, but the forms of aminoacids relevant to living organisms are the zwitterions. Each asymmetric carbongives rise to two optical isomers which are traditionally distinguished by theletters D or L. Only those amino acids which are the L forms (left-handed) atthe alpha carbon are found in terrestrial life. life.

Fig. 27: Amino acids with hydrophobic side groups

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Fig 28 : Amino acids with hydrophilic side groups

Fig. 29 Amino acids for cysteine, proline, tyrosine and tryptophan

Amino acids are the “building Blocks” of the body. Besides building cellsand repairing tissue, they form antibodies to combat invading bacteria &viruses; they are part of the enzyme & hormonal system; they buildnucleoproteins (RNA & DNA); they carry oxygen throughout the body andparticipate in muscle activity. When protein is broken down by digestion theresult is 22 known amino acids. Eight are essential (cannot be manufacturedby the body) the rest are non-essential ( can be manufactured by the bodywith proper nutrition).

6.3 DIFFERENT LEVEL OF ORGANIZATIONA polypeptide chain consists of a linear sequence of peptide linkages with

the remaining R groups of the amino acids branching from the chain like

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leaves from a twig. Although these R groups contain other organic functionalgroups within them, under physiological conditions the different R groups donot generally react with each other to form covalent bonds which link twopolypeptide chains together or form cross-links from one part of a polypeptidechain to another part of the same chain.

A) PRIMARY STRUCTUREThe sequential order of the amino acids that make up its polypeptide

chains, is the most important factor in establishing the three-dimensionalstructure of the protein molecule. One group on one of the common aminoacids—the -SH sulfhydryl group of cysteine—reacts with itself to form disulfidebridges between polypeptide chains. The formation of a cysteine bridge is areduction reaction.

B) SECONDARY STRUCTUREProteins may consist of a single polypeptide chain, as myoglobin does, or

of multiple chains linked by disulfide bonds; the two chains of insulin arejoined by two disulfide bonds. More complex proteins may consist of multiplechains held together by noncovalent forces. Some protein molecules containorganic structures which are not polypeptide chains. Hemoglobin, for example,includes the additional iron-containing heme group which is essential for itstransport of oxygen. The four polypeptide chains of hemoglobin (two of onekind and two of another) are held together by noncovalent forces. The effectof the noncovalent forces, particularly hydrogen bonding, is often to formlocal regions of ordered structures within the protein. One common type oflocal order is the alpha helix structure discovered by the American chemistLinus Pauling.The effects of local ordering, of disulfide bridge formation, andof additional organic structures establish the secondary structure of a protein,which is its overall three-dimensional configuration. The detailed three-dimensional structure of a protein molecule, called its tertiary structure,can be established for many proteins by use of x-ray crystallography. Knowledgeof the tertiary structure is often necessary to understand the chemical andphysiological actions of protein molecules, since their most significant actionsmay involve only a single small active site on a very large molecule.

* PROPERTIES OF THE Á-HELIXThe structure repeats itself every 5.4 Å along the helix axis, i.e. we say

that the á -helix has a pitch of 5.4 Å. á -helices have 3.6 amino acid residuesper turn, i.e. a helix 36 amino acids long would form 10 turns. The separationof residues along the helix axis is 5.4/3.6 or 1.5 Å, i.e. the á -helix has a riseper residue of 1.5 Å. Every main chain C=O and N-H group is hydrogen-bonded to a peptide bond 4 residues away (i.e. Oi to Ni+4). This gives a veryregular, stable arrangement. The peptide planes are roughly parallel withthe helix axis and the dipoles within the helix are aligned, i.e. all C=O groupspoint in the same direction and all N-H groups point the other way. Sidechains point outward from helix axis and are generally oriented towards its

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amino-terminal end. All the amino acids have negative phi and psi angles,typical values being -60 degrees and -50 degrees, respectively.All the aminoacids have negative phi and psi angles, typical values being -60 degrees and -50 degrees, respectively.

Helix

Fig. 30 : Structure of Amino acids

* HELICESStrictly, these form a distinct class of helix but they are always short and

frequently occur at the termini of regular á -helices. The name 310 arisesbecause there are three residues per turn and ten atoms enclosed in a ringformed by each hydrogen bond (note the hydrogen atom is included in thiscount). There are main chain hydrogen bonds between residues separated bythree residues along the chain (i.e. Oi to Ni+3). In this nomenclature thePauling-Corey á -helix is a 3.613-helix. The dipoles of the 310-helix are not sowell aligned as in the á -helix, i.e. it is a less stable structure and side chainpacking is less favourable.

* βββββ-sheet structure. Pauling and Corey derived a model for theconformation of fibrous proteins known as â -keratins. In thisconformation the polypeptide does not form a coil. Instead, it zigzagsin a more extended conformation than the á -helix. â sheets arecompact and stable structures. They are formed when two or morelengths of a protein chain lie next to each other so as to form hydrogenbonds between their respective backbones. In order for the backbonesto be close enough for hydrogen bonds to form, the side chains mustnot come between the backbones. Each length that participates in a âsheet is called a â strand. There are two ways the strands can orientthemselves to form &beta sheets: parallel and anti-parallel.Anti-parallel â Sheets: Protein chains are synthesized starting at the aminoterminus and ending at the carboxyl terminus. Thus a protein chainhas directionality. In an anti-parallel â sheet, the beta strands arealigned next to each other, running in opposite directions.

* REVERSE TURNSA reverse turn is region of the polypeptide having a hydrogen bond from

one main chain carbonyl oxygen to the main chain N-H group 3 residuesalong the chain (i.e. Oi to Ni+3). Helical regions are excluded from this definitionand turns between â -strands form a special class of turn known as the â -hairpin (see later). Reverse turns are very abundant in globular proteins andgenerally occur at the surface of the molecule. It has been suggested that

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turn regions act as nucleation centres during protein folding. Reverse turnsare divided into classes based on the ø and ö angles of the residues at positionsi+1 and i+2.

Fig. 31: Different level of organization

6.4 LET US SUM UPProteins are large organic compounds made of amino acids arranged in a

linear chain and joined together by peptide bonds between the carboxyl andamino groups of adjacent amino acid residues.

An amino acid is by definition an organic compound containing an aminegroup and a carboxylic acid group in the same molecule.

A polypeptide chain consists of a linear sequence of peptide linkages withthe remaining R groups of the amino acids branching from the chain likeleaves from a twig.

Protein structure mainly has two types of organization such as primarystructure and secondary structure.

Primary structure is the linear arrangement of amino acids.

Secondary structure is again divided in to alpha helix and beeta sheets.

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Reverse turns are another type of organization which are formed mainlydue to the hydrogen bonding occurring in the main chain protein structure.

6.5 POINTS FOR DISCUSSION“Proteins and amino acids are the building blocks of organisms” –Justify.

6.6 CHECK YOUR PROGRESSExplain different level of organization of proteins?Note: a) Please don’t proceed till you attempt the above question.


6.7 LESSON-END ACTIVITIES1) What is amino acid and briefly discuss the different types.2) Explain the different level of organization of protein structure3) Define á-helix and â-sheet structure

6.8 REFERENCES





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LESSON - 7

LIPIDS

CONTENTS

7.0 Aims and Objectives7.1 Introduction7.2. Fatty acid7.3 Triacylglycerol (TAG)7.4 Sphingolipids 7.5 Steroid 7.6 Glycerophospholipid 7.7 Let us sum up 7.8 Points for Discussion 7.9 Check your Progress 7.10 Lesson-end activities 7.11 References

7.0 AIMS AND OBJECTIVESTo know about the fatty acids and their functions.

7.1. INTRODUCTIONLipids can be broadly defined as any fat-soluble (hydrophobic) naturally-

occurring molecules. The term is more-specifically used to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides andphospholipids) as well as other fat-soluble sterol-containing metabolites suchas cholesterol. Lipids serve many functions in living organisms includingnutrients, energy storage, structural components of cell membranes, andimportant signaling molecules. Although the term lipid is sometimes used asa synonym for fat, it is in fact a subgroup of lipids called triglycerides andshould not be confused with the term fatty acid.

3.2. FATTY ACIDA fatty acid is a carboxylic acid often with a long unbranched aliphatic tail

(chain), which is either saturated or unsaturated. Carboxylic acids as short asbutyric acid (4 carbon atoms) are considered to be fatty acids, while fatty acidsderived from natural fats and oils may be assumed to have at least 8 carbonatoms, e.g. caprylic acid (octanoic acid). Most of the natural fatty acids have aneven number of carbon atoms, because their biosynthesis involves acetyl-CoA,a coenzyme carrying a two-carbon-atom group (see fatty acid synthesis).

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Saturated fatty acids do not contain any double bonds or other functionalgroups along the chain. The term “saturated” refers to hydrogen, in that allcarbons (apart from the carboxylic acid [-COOH] group) contain as manyhydrogens as possible. In other words, the omega (ù) end contains 3 hydrogens(CH3-) and each carbon within the chain contains 2 hydrogen

Unsaturated fatty acids are of similar form, except that one or morealkenyl functional groups exist along the chain, with each alkene substitutinga singly-bonded “ -CH2-CH2-” part of the chain with a doubly-bonded “-CH=CH-” portion (that is, a carbon double bonded to another carbon).

Essential fatty acids are polyunsaturated fatty acids and are the parentcompounds of the omega-6 and omega-3 fatty acid series, respectively. Theyare essential in the human diet because there is no synthetic mechanism forthem. Humans can easily make saturated fatty acids or monounsaturatedfatty acids with a double bond at the omega-9 position, but do not have theenzymes necessary to introduce a double bond at the omega-3 or omega-6position.

Free fatty acids: Fatty acids can be bound or attached to other molecules,such as in triglycerides or phospholipids. When they are not attached toother molecules, they are known as “free” fatty acids. The uncombined fattyacids or free fatty acids may come from the breakdown of a triglyceride intoits components (fatty acids and glycerol).Free fatty acids are an importantsource of fuel for many tissues since they can yield relatively large quantitiesof ATP. Many cell types can use either glucose or fatty acids for this purpose.In particular, heart and skeletal muscle prefer fatty acids. The brain cannotuse fatty acids as a source of fuel; it relies on glucose, or on ketone bodies.Ketone bodies are produced in the liver by fatty acid metabolism duringstarvation, or during periods of low carbohydrate intake.

A trans fatty acid (commonly shortened to trans fat) is an unsaturatedfatty acid molecule that contains a trans double bond between carbon atoms,which makes the molecule less ‘kinked’ in comparison to fatty acids with cisdouble bonds. These bonds are characteristically produced during industrialhydrogenation of plant oils. Research suggests that amounts of trans fatscorrelate with circulatory diseases such as atherosclerosis and coronary heartdisease more than the same amount of non-trans fats, for reasons that arenot well understood

7.3 TRIACYLGLYCEROL (TAG)TAGs are storage lipids stored mostly in adipose (fat) cells and tissues,

which are highly concentrated stores of metabolic energy. As the termtriacylglycerols implies the molecules are composed of three FA attached to aglycerol skeleton. TAG are excellent storage forms of energy because of thehigh number of reduced CH groups available for oxidation dependent energygeneration processes.The average male (65 kg) stores approx. 420,000 kJ as

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TAG, and only 2500 kJ as glycogen. TAG is the preferred form for energystorage because FA have greater potential chemical energy due to the reducedCH groups than does glucose, which is already partly oxidised with plenty ofOH groups. Thus oxidation of FA yields 40 kJ/g whereas oxidation of glucoseyield 18 kJ/g. Also TAG provide a more efficient form of storage because theyare almost completely anhydrous with no bound water.

Glycogen is very polar and binds water molecules such that 1g of glycogenbinds 2g water. Thus 100g of glycogen stored in the liver is actually 33gglycogen and 66g water, while 100g of TAG in adipose cells is 100g of TAG. Ifthe average male were to store the 420,000 kJ as glycogen then in order toaccommodate the water he would need to weigh 125 kg (21 stone).

TAG’s are formed when ester bonds are formed between the three OHgroups of glycerol and the acid carboxyl groups of three FA. The three FA arenormally different e.g.1-palmitoyl-2-palmitoleol-3-steroyl glycerol containspalmitate, palmitoleate and stearate.When bound in TAG, the FA lose theionised carboxyl group so are not charged thus TAG are also called neutralfats. The loss of the polar head makes the entire molecule very hydrophobic,and almost completely insoluble in water so that TAG are stored as oil dropletsin adipose cells. Almost always find two saturated FA with one unsaturatedFA in TAG, because 3 saturated FA would provide TAG that was solid wax-type at room temperature. More than one unsaturated FA, and the TAG wouldbe too fluid for storage purposes inside cells.

7.4 SPHINGOLIPIDSThese are a class of lipids derived from the aliphatic amino alcohol

sphingosine. Sphingolipids are often found in neural tissue, and play animportant role in both signal transmission and cell recognition.The sphingosinebackbone is O-linked to a (usually) charged head group such as ethanolamine,serine, or choline.The backbone is also amide-linked to an acyl group, suchas a fatty acid.There are three main types of sphingolipids:

Ceramides. Ceramides are the simplest type of sphingolipid. They consistsimply of a fatty acid chain attached through an amide linkage to sphingosine.

Sphingomyelins. Sphingomyelins have a phosphorylcholine orphosphoroethanolamine molecule esterified to the 1-hydroxy group of aceramide.

Glycosphingolipids, which differ in the substituents on their head group(see image). Glycosphingolipids are ceramides with one or more sugar residuesjoined in a â-glycosidic linkage at the 1-hydroxyl position. Glycosphingolipids may be furthersubdivided into cerebrosides and gangliosides.

Cerebrosides have a single glucose or galactose at the 1-hydroxy position.

Gangliosides have at least three sugars, one of which must be sialic acid.

Function

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Sphingolipids are commonly believed to protect the cell surface againstharmful environmental factors by forming a mechanically stable and chemicallyresistant outer leaflet of the plasma membrane lipid bilayer. Certain complexglycosphingolipids were found to be involved in specific functions, such ascell recognition and signaling. The first feature depends mainly on the physicalproperties of the sphingolipids, whereas signaling involves specific interactionsof the glycan structures of glycosphingolipids with similar lipids present onneighboring cells or with proteins.

7.5 STEROID

Fig. 32 : Steroid skeleton of lanosterol

The total number of carbons (30) reflects its triterpenoid origin. In somesteroids some carbons may be removed (such as carbon 18) or added (such ascarbons 241 and 242) in downstream biosynthetic reactions. A steroid is aterpenoid lipid characterized by a carbon skeleton with four fused rings,generally arranged in a 6-6-6-5 fashion. Steroids can vary by the functionalgroups attached to these rings and the oxidation state of the rings. Hundredsof distinct steroids are found in plants, animals, and fungi. All steroids arebiosynthetically derived either from the sterol lanosterol (animals and fungi)or the sterol cycloartenol (plants). Both sterols are derived from the cyclizationof the triterpene squalene.

Some of the common categories of steroids:Animal steroidsInsect steroids Ecdysteroids such as ecdysteroneVertebrate steroids Steroid hormonesSex steroids -androgens, estrogens, and progestagens.Corticosteroids - glucocorticoids and mineralocorticoids. GlucocorticoidsAnabolic steroids -CholesterolPlant steroids -Phytosterols ,BrassinosteroidsFungus steroids –Ergosterols

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7.6 GLYCEROPHOSPHOLIPID

Fig. 33 : Glycerol

Glycerophospholipids or phosphoglycerides are glycerol-basedphospholipids. They are the main component of biological membranes.Theterm glycerophospholipid signifies any derivative of sn-glycero-3-phosphoricacid that contains at least one O-acyl, or O-alkyl or O-alk-1'-enyl residueattached to the glycerol moiety and a polar head made of a nitrogenous base,a glycerol, or an inositol unit.It contains a glycerol core with fatty acids. Theycan be the same or different subunits of fatty acids.

• Carbon 1 (tail, apolar) contains a fatty acid, typically saturated• Carbon 2 (tail, apolar) contains a fatty acid, typically unsaturated and

in the cis conformation, thus appearing “bent”• Carbon 3 (head, polar) contains a phosphate group or an alcohol

attached to a phosphate group

Lecithin and cephalin are more common than the others in most humanmembranes, but cardiolipin is quite common in the inner membranes ofmitochondria. One of a glycerophospholipid’s functions is to serve as astructural component of cell membranes. The cell membrane seen under theelectron microscope consists of two identifiable layers, or “leaflets”, each ofwhich is made up of an ordered row of glycerophospholipid molecules. Thecomposition of each layer can vary widely depending on the type ofcell.Glycerophospholipids can also act as an emulsifying agent to promotedispersal of one substance into another. This is sometimes used in candymaking.

7.7 LET US SUM UPLipids are those macromolecules which has a wide variety of fatty acids

and their derivatives.

Lipids serve many functions in living organisms including nutrients, energystorage, structural components of cell membranes, and important signalingmolecules.

A fatty acid is a carboxylic acid often with a long unbranched aliphatictail (chain), which is either saturated or unsaturated.

Saturated fatty acids do not contain any double bonds or other functionalgroups along the chain.

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Triacyl Glycerides are storage lipids stored mostly in adipose (fat) cellsand tissues, which are highly concentrated stores of metabolic energy.

TAG’s are formed when ester bonds are formed between the three OHgroups of glycerol and the acid carboxyl groups of three fatty acids.

Sphingolipids are a class of lipids derived from the aliphatic amino alcoholsphingosine.

A steroid is a terpenoid lipid characterized by a carbon skeleton with fourfused rings, generally arranged in a 6-6-6-5 fashion.

Glycerophospholipids or phosphoglycerides are glycerol-basedphospholipids. They are the main component of biological membranes.

7.8 POINTS FOR DISCUSSIONMake a complete analysis of lipids as macromolecules.

7.9 CHECK YOUR PROGRESSDescribe a few points on steroids?Note: a) Please don’t proceed till you attempt the above question.


7.10 LESSON-END ACTIVITIES1) Draw the chemical structures of Fatty acids2) Define Steroid and Glycerophospholipid3) What are Triacylglycerol (TAG) and Sphingolipids.






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LESSON - 8

NUCLEIC ACIDS

CONTENTS


8.1 Introduction

Purine

Pyrimidine

Nucleoside

Nucleotide

8.2. Different forms of DNA

8.3. Let us sum up

8.4. Points for Discussion

8.5. Check your Progress

8.6. Lesson-end activities

8.7. References

8.0 AIMS AND OBJECTIVESTo learnt he concepts like purine, pyrimidine and forms of DNA.

Also the denaturation and renaturation of proteins are discussed.

8.1. INTRODUCTIONLiving organisms are complex systems. Hundreds of thousands of proteins

exist inside each one of us to help carry out our daily functions. These proteinsare produced locally, assembled piece-by-piece to exact specifications. Anenormous amount of information is required to manage this complex systemcorrectly. This information, detailing the specific structure of the proteinsinside of our bodies, is stored in a set of molecules called nucleic acids. Thenucleic acids are very large molecules that have two main parts. The backboneof a nucleic acid is made of alternating sugar and phosphate molecules bondedtogether in a long chain, represented below.

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Fig. 34 : Purines

Purine: It is a heterocyclic aromatic organic compound,consisting of apyrimidine ring fused to an imidazole ring. Purines make up one of the twogroups of nitrogenous bases. Pyrimidines make up the other group. Thesebases make up a crucial part of both deoxyribonucleotides and ribonucleotides,and the basis for the universal genetic code

Pyrimidine: is a heterocyclic aromatic organic compound similar to benzeneand pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring.[1] It is isomeric with two other forms of diazine

Fig. 35 Pyrimidine

Nucleoside: Purine or pyrimidine base linked glycosidically to ribose ordeoxyribose, but lacking the phosphate residues that would make it anucleotide.

Nucleotide: Phosphate esters of nucleosides. The metabolic precursorsof nucleic acids are monoesters with phosphate on carbon 5 of the pentose(known as 5' to distinguish sugar from base numbering).DNA is basically along molecule that contains coded instructions for the cells. Everything thecells do is coded somehow in DNA - which cells should grow and when, whichcells should die and when, which cells should make hair and what color itshould be. Our DNA is inherited from our parents. We resemble our parentssimply because our bodies were formed using DNA to guide the process - theDNA we inherited from them.

DNA is a long molecule, like a chain, where the links of the chain arepieces called nucleotides (sometimes also called ‘bases’). There are fourdifferent types of nucleotides in DNA which we’ll call ‘A’, ‘G’, ‘C’ and ‘T’. These

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four are all that’s necessary to write a code that describes our entire bodyplan.

The four nucleotides look a little bit alike. They all have a ring of carbonscalled, in chemist’s terminology, a ‘sugar’ (not the same as ‘table sugar’,however). Each nucleotide also has another type of ring structure, and this iswhere the four types of nucleotide are different. These rings are organicbases, much like the more familiar mineral acids and bases like NaOH orHCl, except these bases are composed of carbon, nitrogen and oxygen.

DNA chains are made by connecting those nucleotides together viachemical bonds. At right is a diagram showing four nucleotides connected toform an oligonucleotide, in this case an RNA oligo (note that it has ‘-OH’ atthe lower right corner of each nucleotide, as opposed to the ‘-H’ in DNA). I’veleft off the bases, for simplicity’s sake. You can see the sugar rings linkedtogether with phosphate bridges. This is a “single-stranded” nucleic acid.Below is the double-stranded form.Double-stranded DNA is simply two chainsof single- stranded DNA, positioned so their “bases” can interact with eachother. At left is a cartoon depiction of double-stranded DNA.

Importantly, the two strands travel in opposite directions; hence thestructure is said to be “anti-parallel”.The bases in the middle “pair up” withbases on the opposite strand, so that a type ‘A’ nucleotide is always opposite atype ‘T’, and ‘G’ is opposite ‘C’. The attraction between the paired nucleotidesis fairly weak, but when there is a whole string of them, it adds up to enoughstrength to hold the strands together.

8.2. DIFFERENT FORMS OF DNAThere are three natural forms of DNA (A, B and Z). The origin of these

different forms are related to the conformation of the sugar (C2'-endo/ C3'-endo) and the orientation of the base relative to the sugar (syn/anti).Thusdepending on base composition and physical conditions (Hydration/Salt-Content), DNA can assume several different conformations (A, B, Z).Eachconformation possesses specific parameters: diameter of the helix, number ofbases per tour and distance between plan of bases.

The B-form is the common natural form, prevailing under physiologicalconditions of low ionic strength and high degree of hydration. B-DNA arranges10 nucleotides per helix tour, all of conformation C2'-endo/anti . The plane ofthe bases is nearly perpendicular to the helix axis and the helix surfaceexhibits two prominent grooves (major and minor).

The Z-form (Zigzag chain) is observed in DNA G-C rich local region. Z-DNA is longer, thinner and possess an unusual left-handed helix (of 12 basespairs/tour) with a single narrow deep groove. These Zigzag form mainly resultsfrom the alternation of purines (C3'-endo/syn) and pyrimidines (C2'-endo/anti).

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The A-form is sometimes found in some parts of natural DNA in presenceof high concentration of cations or at a lower degree of hydration (<65%). A-DNA possess 11 nucleotides per tour (all C3'-endo/anti) and two grooves (anarrow deep major and a wide shallow minor).

The C-form and D-form are unusual subclasses of B-type. C-DNA issometimes observed under 45% of hydration while D-DNA is only found inartificial DNA.

The changes in the shape of DNA can affect its binding with proteins andmay be involved in some regulation process during replication or transcription.

8.3 LET US SUM UPNucleic acids are large molecules which has the genetic code of organisms.

The backbone of a nucleic acid is made of alternating sugar and phosphatemolecules bonded together in a long chain. Nucleic acids contain nitrogenousbases, sugar, and a phosphate residue. DNA is a long molecule, like a chain,where the links of the chain are pieces called nucleotides. Dna contains thefollowing nucleotides A,C,G, and T. DNA mainly has three natural forms likeA,B and Z. The conversion of double stranded DNA to single stranded is knownas denaturation. The conversion of single strands back to the double-strandedstructure is called renaturation.

8.4 POINTS FOR DISCUSSIONDo a closer analysis on the functions of purines and pyrimidines.

8.5 CHECK YOUR PROGRESSExplain the different forms of DNA?Note: a) Please don’t proceed till you attempt the above question.


8.6 LESSON-END ACTIVITIES1) Give the structures of purines and pyrimidines2) Define nucleotide and Nucleoside3) What are the different forms of DNA.4) Explain the denaturation of Nucleic Acids & renaturation

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8.7 REFERENCES





65

UNIT III

66

67

LESSON - 9

CELL ENERGETICS

CONTENTS


9.1 Cell energetics

9.1.1 Glycolysis

9.1.2 Aerobic oxidation

9.1.3 Photosynthesis

9.2. Let us sum up

9.3. Points for Discussion

9.4. Check your Progress

9.5. Lesson-end activities

9.6. References

9.0 AIMS AND OBJECTIVESTo know the steps in glycolysis and photosynthesis.

9.1. CELL ENERGETICSThe most important molecule for capturing and transferring free energy

in biological systems is adenosine triphosphate, or ATP. Under standardconditions, hydrolysis of the terminal high-energy phosphoanhydride bond inATP to yield adenosine diphosphate (ADP) and inorganic phosphate (Pi) releases7.3 kcal/mol of free energy. Cells can use the energy released during thisreaction to power many otherwise energetically unfavorable processes, suchas the transport of molecules against a concentration gradient by ATP-poweredpumps the movement (beating) of cilia, the contraction of muscle (and thesynthesis of proteins from amino acids and of nucleic acids from nucleotides).Although other high-energy molecules occur in cells, ATP is the universal“currency” of chemical energy; it is found in all types of organisms and musthave occurred in the earliest life-forms.

This chapter focuses on how cells generate the high-energyphosphoanhydride bond of ATP from ADP and Pi. This endergonic reaction,which is the reverse of ATP hydrolysis and requires an input of 7.3 kcal/mol

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to proceed, where Pi2" represents inorganic phosphate (HPO42"). The energyto drive this reaction is produced primarily by two main processes — aerobicoxidation, which occurs in nearly all cells, and photosynthesis, which occursonly in leaf cells of plants and certain single-celled organisms.

9.1.1 GLYCOLYSIS

PATHWAYThe most common and well-known type of glycolysis is the Embden-

Meyerhof pathway. Glycolysis is a metabolic pathway by which a 6-carbonglucose (Glc) molecule is oxidized to two molecules of pyruvic acid (Pyr). Theword glycolysis is derived from Greek ãëõêýò (sweet) and ëýóéò (rupture). Itis the initial process of most carbohydrate catabolism, and it serves threeprincipal functions:

Fig. 36: Pathway of Glycolysis

Fig. 37 : Pathway of Glycolysis

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• The generation of high-energy molecules (ATP and NADH) as cellularenergy sources as part of anaerobic and aerobic respiration.

• Production of pyruvate for the citric acid cycle as part of aerobicrespiration.

• The production of a variety of six- and three-carbon intermediatecompounds, which may be removed at various steps in the process forother cellular purposes.

As the foundation of both aerobic and anaerobic respiration, glycolysis isthe archetype of universal metabolic processes known and occurring (withvariations) in many types of cells in nearly all organisms. Glycolysis, throughanaerobic respiration, is the main energy source in many prokaryotes,eukaryotic cells devoid of mitochondria (e.g. mature erythrocytes) andeukaryotic cells under low oxygen conditions (e.g. heavily exercising muscleor fermenting yeast).In eukaryotes and prokaryotes, glycolysis takes placewithin the cytosol of the cell. In plant cells some of the glycolytic reactionsare also found in the Calvin-Benson cycle which functions inside thechloroplasts. The wide conservation includes the most phylogenetically deeprooted extant organisms and thus it is considered to be one of the mostancient metabolic pathways.

REGULATION OF GLYCOLYSISKey enzymes: Glucokinase, Phosphofructokinase-1 & Pyruvate kinase.Insulin stimulates the synthesis of key enzymes responsible forglycolysis.The hormones epinephrine & glucagon increase cAMP level to activatecAMP-dependent protein kinase which phosphorylates & inactivatespyruvate kinase & inhibit glycolysis.Phosphofructo kinase is involved in feed back control.

Inhibitors: Iodoacetate, arsenite & fluoride.

9.1.2 AEROBIC OXIDATIONIn aerobic oxidation, fatty acids and sugars, principally glucose, are

metabolized to CO2 and H2O, and the released energy is converted to thechemical energy of phosphoanhydride bonds in ATP. In animal cells and mostother nonphotosynthetic cells, ATP is generated mainly by this process. Theinitial steps in the oxidation of glucose, called glycolysis, occur in the cytosolin both eukaryotes and prokaryotes and do not require O2. The final steps,which require O2, generate most of the ATP. In eukaryotes, the later stagesof aerobic oxidation occur in mitochondria, whereas in prokaryotes, whichlack mitochondria, many of the final steps occur on the plasma membrane.The final stages of fatty acid metabolism sometimes occur in mitochondriaand generate ATP; in most eukaryotic cells, however, fatty acids aremetabolized in peroxisomes without production of ATP.

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9.1.3 PHOTOSYNTHESISIn photosynthesis, light energy is converted to the chemical energy of

phosphoanhydride bonds in ATP and stored in the chemical bonds ofcarbohydrates (primarily sucrose and starch). Oxygen also is formed duringphotosynthesis. In plants and eukaryotic single-celled algae, photosynthesisoccurs in chloroplasts. Although they lack chloroplasts, several prokaryotesalso carry out photosynthesis by a mechanism similar to that in chloroplasts.The oxygen generated during photosynthesis is the source of virtually all theoxygen in the air, and the carbohydrates produced are the ultimate source ofenergy for virtually all nonphotosynthetic organisms.

At first glance, photosynthesis and aerobic oxidation appear to have littlein common. However, a revolutionary discovery in cell biology is that bacteria,mitochondria, and chloroplasts all use the same (or very nearly the same)process, called chemiosmosis (or chemiosmotic coupling), to generate ATPfrom ADP and Pi . The immediate energy sources that power ATP synthesisare the transmembrane proton concentration gradient and electric potential(voltage gradient), collectively termed the proton-motive force. The proton-motive force is generated by the stepwise movement of electrons from higherto lower energy states via membrane-bound electron carriers. In mitochondriaand nonphotosynthetic bacterial cells, electrons from NADH (produced duringthe metabolism of sugars, fatty acids, and other substances) are transferredto O2, the ultimate electron acceptor. In the thylakoid membrane ofchloroplasts, energy absorbed from light strips electrons from water (formingO2) and powers their movement to other electron carriers, particularly NADP+;eventually these electrons are donated to CO2 to synthesize carbohydrates.All these systems, however, contain some similar carriers that couple electrontransport to the pumping of protons (always from the cytosolic face to theexoplasmic face of the membrane), thereby generating the proton-motive force.

Moreover, all cells utilize essentially the same kind of membrane protein,the F0F1 complex, to synthesize ATP. The F0F1 complex, also called ATPsynthase and F0F1 ATPase, is a member of the F class of ATP-powered protonpumps . In all cases, the F0F1 complex is positioned with the globular F1segment, which catalyzes ATP synthesis, on the cytosolic face of the membrane,so ATP is always formed on the cytosolic face of the membrane .(Protonsalways flow through the F0F1 complex from the exoplasmic to the cytosolicface of the membrane, driven by a combination of the proton concentrationgradient (exoplasmic face > cytosolic face) and the membrane electric potential(exoplasmic face positive with respect to the cytosolic face).

In addition to powering ATP synthesis, the proton-motive force can supplyenergy for the transport of small molecules across a membrane against aconcentration gradient example, the uptake of lactose by certain bacteria iscatalyzed by a H+/sugar symport protein, and the accumulation of ions andsucrose by plant vacuoles is catalyzed by proton-driven antiporters. The rotationof bacterial flagella is also powered by the proton-motive force; in contrast,

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the beating of eukaryotic cilia is powered by ATP hydrolysis. Conversely,hydrolysis of ATP by V-class ATP-powered proton pumps, which are similar instructure to P-class pumps ,provides the energy for transporting protons againsta concentration gradient. Chemiosmotic coupling thus illustrates an importantprinciple introduced in our discussion of active transport in: the membranepotential, the concentration gradients of protons (and other ions) across amembrane, and the phosphoanhydride bonds in ATP are equivalent andinterconvertible forms of chemical potential energy.

9.2 LET US SUM UPThe most important molecule for capturing and transferring free energy

in biological systems is adenosine triphosphate, or ATP.

The energy to drive this reaction is produced primarily by aerobic oxidation,and photosynthesis.

Glucokinase, Phosphofructokinase-1 & Pyruvate kinase are the keyenzymes for glycolysis.

Iodoacetate, arsenite & fluoride are the inhibitors for glycolysis.

9. 3 POINTS FOR DISCUSSION“Glycolysis is the formulation for both aerobic and anaerobic respiration”

– Substantiate.

9.4 LESSON-END ACTIVITIESDescribe the Cell energetics and Glycolysis.

What is photosynthesis?

9.5 CHECK YOUR PROGRESSGive an account of anaerobic respiration. Give an Example.Note: a) Please don’t proceed till you attempt the above question.


9.6 REFERENCES


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73

LESSON - 10

UTILIZATION OF GLUCOSE, FAT AND PROTEIN

CONTENTS


10.1 Introduction

10.2 Let us Sum Up



10.5 Lesson-End Activities

10.6 References

10.0 AIMS AND OBJECTIVESAs already mentioned, metabolism refers to the chemical reactions carried

out inside of the cell. The major metabolic reactions which we will study arethose involving catabolism which is the breakdown of larger molecules toextract energy. We will focus our discussion on the individual steps in themetabolic reactions where energy is produced. Some attention will also begiven to the synthesis of other biomolecules.

The overall reaction for the combustion of glucose is written:C6H12O6 + 6 O2 ——> 6 CO2 + 6 H2O + energy

10.1 INTRODUCTIONAlthough the above equation represents the overall metabolic reaction for

carbohydrates, there are actually over thirty individual reactions. Eachreaction is controlled by a different enzyme. The failure of an enzyme tofunction may have serious and possibly fatal consequences. Slightly less thanhalf of the 686 kcal/mole of the energy produced by combustion is availablefor storage and use by the cell with the remaining amount dissipated as heat.

Metabolism will be studied in various parts. Interrelationships will bepointed out as they are encountered. Just as there are three basic biomolecules- carbohydrates, lipids, and proteins, the metabolism of each of these will bestudied individually. The interrelationships of the major components inmetabolism are diagramed in Figure 1. At the end of the study of metabolism,you may be asked to diagram portions of it from memory. A complete dietmust supply the elements; carbon, hydrogen, oxygen, nitrogen, phosphorus,

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sulfur, and at least 18 other inorganic elements. The major elements aresupplied in carbohydrates, lipids, and protein. In addition, at least 17 vitaminsand water are necessary. If an essential nutrient is omitted from the diet,certain deficiency symptoms appear.

Carbohydrates: Foods supply carbohydrates in three forms: starch, sugar,and cellulose (fiber). Starch and sugar are major and essential sources ofenergy for humans. A lack of carbohydrates in the diet would probably resultin an insufficient number of calories in the diet. Cellulose furnishes bulk inthe diet.

Since the tissues of the body need glucose at all times, the diet mustcontain substances such as carbohydrates or substances which will yieldglucose by digestion or metabolism. For the majority of the people in theworld, more than half of the diet consists of carbohydrates from rice, wheat,bread, potatoes, macaroni.

Proteins: All life requires protein since it is the chief tissue builder andpart of every cell in the body. Among other functions, proteins help to: makehemoglobin in the blood that carries oxygen to the cells; form anti-bodies thatfight infection; supply nitrogen for DNA and RNA genetic material; and supplyenergy.

Proteins are necessary for nutrition because they contain amino acids.Among the 20 or more amino acids, the human body is unable to synthesize8, therefore, these amino acids are called essential amino acids. A foodcontaining protein may be of poor biological value if it is deficient in one ormore of the 8 essential amino acids: lysine, tryptophan, methionine, leucine,isoleucine, phenylalanine, valine, and threonine. Proteins of animal originhave the highest biological value because they contain a greater amount ofthe essential amino acids. Foods with the best quality protein are listed indiminishing quality order: whole eggs, milk, soybeans, meats, vegetables,and grains.

Fats and Lipids: Fats are concentrated sources of energy because theygive twice as much energy as either carbohydrates or protein on a weightbasis. The functions of fats are to: make up part of the structure of cells, forma protective cushion and heat insulation around vital organs, carry fat solublevitamins, and provide a reserve storage for energy. Three unsaturated fattyacids which are essential include: linoleic, linolinic, and arachidonic andhave 2, 3, and 4 double bonds respectively. Saturated fats, along withcholesterol, have been implicated in arteriosclerosis, “hardening of thearteries”. For this reason, the diet should be decreased in saturated fats(animal) and increased in unsaturated fat (vegetable).

a) MH + NAD+ —> NADH + H+ + M + energyb) ADP + P + energy —> ATP + H2O

The major metabolic reactions which we will study are those involving

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catabolism which is the breakdown of larger molecules to extract energy. Wewill focus our discussion on the individual steps in the metabolic reactionswhere energy is produced. Some attention will also be given to the synthesisof other biomolecules. The overall reaction for the combustion of glucose iswritten:

C6H12O6 + 6 O2 ——> 6 CO2 + 6 H2O + energy

Although the above equation represents the overall metabolic reaction forcarbohydrates, there are actually over thirty individual reactions. Eachreaction is controlled by a different enzyme. The failure of an enzyme tofunction may have serious and possibly fatal consequences.

10.2 LET US SUM UPFoods supply carbohydrates in three forms: starch, sugar, and cellulose

(fiber).

All life requires protein since it is the chief tissue builder and part ofevery cell in the body.

Fats are concentrated sources of energy because they give twice as muchenergy as either carbohydrates or protein on a weight basis.

10.3 POINTS FOR DISCUSSIONHow glucose, fat and protein are effectively used within our body? Do an

analysis.

10.4 CHECK YOUR PROGRESSGive an account of anaerobic respiration. Give an Example.Note: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer.

10.5 LESSON-END ACTIVITIES1. How the lipid molecules are getting utilized in the body?2. Define carbohydrate, proteins, fats and lipids.............................................................................................................................................................................................................................................................................................................................................................................................................................................................

10.6 REFERENCES


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77

UNIT IV

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79

LESSON - 11

ENZYMES

CONTENTS


11.1 Enzymes -Unit of Activity

11.2 Coenzymes And Metal Cofactors

11.3 Factors affecting enzymatic activity

11.4 Let us sum up




11.8 References

11.0 AIMS AND OBJECTIVESTo know and understand the different enzymes and factors affecting

enzymatic activity.

11.1 ENZYMES -UNIT OF ACTIVITYAn enzyme is a protein made up of a sequence of the twenty amino acids.

Its chemical properties are effectively limited to those available from thatlimited number of building blocks. Enzymes are globular proteins - theirmolecules are round in shape. They have an area - usually thought of as apocket-shaped gap in the molecule - which is called the active site. Someenzymes are found inside cells (intracellular enzymes), and some - especiallydigestive enzymes - are released so they have their effects outside the cell(extracellular enzymes). Only the substrate (or substrates) fits/fit into theactive site. There are several types of enzyme which contribute to differenttypes of biochemical reaction.

Fig. Biochemical reactions

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The enzyme speeds up the process of conversion of substrates (reactants)into products - usually so much that the reaction does not take place in theabsence of enzyme.

Although the enzyme obviously joins with the substrate for a short while,the enzyme and substrate split apart afterwards, releasing the enzyme. Thusthe enzyme is not used up in the process (unlike the substrate(s)), so it cancontinue to react if more substrate is provided.

11.2 COENZYMES AND METAL COFACTORSCoenzymes are small organic non-protein molecules that carry chemical

groups between enzymes. Many enzymes require additional help in catalysingtheir reaction from a coenzyme or cofactor. This is a small organic molecule,or a metallic ion, which carry out some part of the catalytic process beyondthe chemical abilities of the enzyme itself. Coenzymes can fill any gaps inthe chemical armory of proteins by introducing other types of chemicalstructures.

The name “coenzyme” suggests that a molecule has catalytic propertiesused in cooperation with the enzyme proper,speed up the reaction but wasitself unchanged .This is certainly true of some coenzymes but othercompounds, frequently referred to as coenzymes, certainly undergo change.Examples of these are ATP (adenosine triphosphate) which is changed to ADP(adenosine diphosphate) in many reactions and NAD (nicotinamide adeninedinucleotide) which is changed to NADH (the reduced form of NAD). Therationale behind referring to these compounds (which are really substratesof the enzymes which use them) as coenzymes is that they are recycled byother metabolic reactions which convert them back to the original compoundagain. Although these coenzymes are changed by an individual enzymereaction, and so are not truly catalytic, they are not permanently changed inmetabolism. They can therefore be regarded as metabolically catalytic. Manycoenzymes are phosphorylated water-soluble vitamins and are also commonlymade from nucleotides such as adenosine triphosphate, the biochemicalcarrier of phosphate groups, or coenzyme A, the coenzyme that carries acylgroups; eg., Vitamin and nucleotide derivatives like Ascorbic acid (Vitamin C); Coenzyme A - Contains pantothenic acid (Vitamin B5) and ATP ; CoenzymeB12 ; Riboflavin (B2) - FAD and FMN ; Thiamine pyrophosphate (B1) ; NAD andNADP - Contain both a nucleotide and a Niacin (vitaimin B3) moiety.

COFACTORA cofactor is a non-protein chemical compound that is bound tightly to an

enzyme and is required for catalysis. They can be considered “helpermolecules/ions” that assist in biochemical transformations. Certain substancessuch as water and various abundant ions may be bound tightly by enzymes,but are not considered to be cofactors since they are ubiquitous and rarelylimiting. An enzyme without a cofactor is referred to as an apoenzyme, and

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the completely active enzyme (in addition to the cofactor) is called aholoenzyme.

Apoenzyme + cofactor <=> Holoenzyme.

Metal ions are common cofactors. In nutrition, the list of essential traceelements reflects their role as cofactors. This list includes manganese, iron,cobalt, nickel, copper, zinc, and molybdenum. Other organisms requireadditional metals, such as vanadium and tungsten. The study of these cofactorsfalls under the area of bioinorganic chemistry. Some cofactors undergochemical changes during the course of a reaction, undergoing reduction oroxidation. Nonetheless, as a catalyst, cofactors return to their original statein the course of the catalytic cycle. They are not consumed. In this respect,cofactors differ from substrates or coenzymes. In many cases, the cofactorincludes both an inorganic and organic components. One diverse set of examplesare the heme proteins, which consists of a porphyrin ring coordinated to iron.Cofactors vary in their location and the tightness of their binding to the hostenzyme. When bound tightly to the enzyme, cofactors are called prostheticgroups. Loosely-bound cofactors typically associate in a similar fashion toenzyme substrates. These are better described as coenzymes, which areorganic substances that directly participate as substrates in an enzymereaction. Vitamins can serve as precursors to coenzymes (e.g. vitamins B1,B2, B6, B12, niacin, folic acid) or as coenzymes themselves (e.g. vitamin C).

11.3 FACTORS AFFECTING ENZYMATIC ACTIVITYWithin the normal range, changes in temperature, pH, and concentrations

of substrate and enzyme affect the rate of reaction in accordance withpredictable interactions between enzyme and substrate molecules.

Effect of temperature:- The effects of temperature may be explained onthe basis of kinetic theory - increased temperature increases the speed ofmolecular movement and thus the chances of molecular collisions, so withina narrow range (often 0-45°C), the rate of reaction is proportional to thetemperature. It is often said that an enzyme’s rate of reaction doubles forevery 10°C rise in temperature. However, the interaction between this positiveeffect of increased temperature and the negative effect results in a differentsituation, so that enzymes may be said to have an optimum temperature fortheir action. Like most chemical reactions, the rate of an enzyme-catalyzedreaction increases as the temperature is raised. A 10°C rise in temperaturewill increase the activity of most enzymes by 50 to 100%. Variations in reactiontemperature as small as 1 or 2 degrees may introduce changes of 10 to 20%in the results. In the case of enzymatic reactions, this is complicated by thefact that many enzymes are adversely affected by high temperatures. Asshown in figure, the reaction rate increases with temperature to a maximumlevel, then abruptly declines with further increase of temperature. Becausemost animal enzymes rapidly become denatured at temperatures above 40°C,most enzyme determinations are carried out somewhat below that temperature.

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Over a period of time, enzymes will be deactivated at even moderatetemperatures. Storage of enzymes at 5°C or below is generally the mostsuitable. Some enzymes lose their activity when frozen.

Effects of pH: Changes in the pH probably affect the attraction betweenthe substrate and enzyme, and thus the efficiency of the conversion process.Often, there is an optimum pH - near to pH 7 (neutral) in intracellular enzymes,and either in the acidic range (perhaps pH 1- 6) or in the alkaline range (pH8-14) for different digestive enzymes. The most favorable pH value - the pointwhere the enzyme is most active - is known as the optimum pH. Extremelyhigh or low pH values generally result in complete loss of activity for mostenzymes. pH is also a factor in the stability of enzymes. As with activity, foreach enzyme there is also a region of pH optimal stability.

Fig. 39 : Graph depicting pH stability

Presence of other substances:- Some enzymes work better if othersubstances are also present. Some enzymes (pepsin - from the stomach) workbetter if acid is present e.g. lipases are more effective if emulsifying agentsare present because they break up the substrate into smaller droplets. Abovenormal temperatures (60 °C), heat alters irreversibly the enzyme molecule.This denaturation is due to molecular vibrations (caused by heat) which changethe shape of the protein, altering the folding and internal cross-linkages inits polypeptide chains. These changes - especially in the region of the activesite - mean that the enzyme is inactivated, even when returned to normaltemperature.

The higher the temperature to which the enzyme is subjected and thelonger the heating is continued, the greater the proportion of damaged enzymemolecules and the result is that the conversion process becomes less andless efficient. Below normal temperatures, enzymes become less and lessactive, due to reductions in speed of molecular movement, but this isreversible, so enzymes work effectively when returned to normal temperature.Enzymes are sometimes adversely affected by other chemical substanceswhich combine with them, either at their active site or by altering the overallshape of their molecule.

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Fig. 40 : Enzyme Reactions

11.4 LET US SUM UPAn enzyme is a protein made up of a sequence of the twenty protein

amino acids.

They have an area - usually thought of as a pocket-shaped gap in themolecule - which is called the active site.

Within the normal range, changes in temperature, pH, and concentrationsof substrate and enzyme affect the rate of reaction in accordance withpredictable interactions between enzyme and substrate molecules. There areapproximately 3000 enzymes which have been characterised.

These are grouped into six main classes according to the type of reactioncatalysed. Such as Oxidoreductases ,Transferases, Hydrolases, Lyases,Isomerases, Ligases.

The mechanism of enzyme action can be described in two ways. First oneis using Lock and Key hypothesis and the second one is Induced fit model.

11.5 POINTS FOR DISCUSSION“The enzyme activity is affected more by the pH than temperature” –

Comment.

11.6 CHECK YOUR PROGRESSGive the effect of pH and temperature on enzymes?Note: a) Please don’t proceed till you attempt the above question.

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11.7 LESSON –END ACTIVITIES1) What are enzymes?2) Explain the unit of activity of the enzymes?3) What are the main factors which affect enzymatic activity?4) Explain the Lock and key model of the active site.5) Explain the Induced fit model of the active site.

11.7 REFERENCES





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LESSON - 12

MICHAELIS MENTON KINETICS

CONTENTS


12.1 Deriving the Michaelis – Menton Equation

12.2 Enzyme Inhibitions

12.3 Enzyme Activators

12.4 Let us Sum Up



12.7 Lesson-end Activities

12.8 References

12.0 AIMS AND OBJECTIVESTo learn to derive Michaelis – Menton equation and to understand the

enzyme inhibitors and activators.

12.1 DERIVING THE MICHAELIS-MENTEN EQUATIONStart with the generalised scheme for enzyme-catalysed production of a

product (P) from substrate (S). The enzyme (E) does not magically convert Sinto P, it must first come into physical contact with it, i.e. E binds S to forman enzyme-substrate complex (ES). Michaelis and Menten therefore set outthe following scheme:

. . . . (1)

The terms k1, k-1 and k2 are rate constants respectively correspondingto the association of substrate and enzyme, the dissociation of unalteredsubstrate from the enzyme and the dissociation of product (= altered substrate)from the enzyme. Note that there is the theoretical possibility of a reversereaction, with ES complex forming from E and P, but this can be ignoredbecause we are considering initial rates of reaction, i.e. when the enzyme isfirst provided with substrate, so there should not be any product available tocombine with enzyme.

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The overall rate of the reaction (v) is limited by the step ES to E + P, andthis will depend on two factors - the rate of that step (i.e. k2) and theconcentration of enzyme that has substrate bound, i.e. [ES]. This can bewritten as:

. . . (2)

At this point it is important to draw your attention to two assumptionsthat are made in this scheme. The first is the availability of a vast excess ofsubstrate, so that [S]>>[E]. Secondly, it is assumed that the system is insteady-state, i.e. that the ES complex is being formed and broken down at thesame rate, so that overall [ES] is constant. The formation of ES will dependon the rate constant K1 and the availability of enzyme and substrate, i.e. [E]and [S]. The breakdown of [ES] can occur in two ways, either the conversionof substrate to product or the non-reactive dissociation of substrate from thecomplex. In both instances the [ES] will be significant. Thus, at steady statewe can write:

. . . (3)

The next couple of steps are rearrangements of this equation. First of allwe can collect together the rate constants on the right-hand side becausethey are both multiplied by [ES], this gives us:

. . . (4)

Then dividing both sides by (k-1 + k2), this becomes:

. . . (5)

Note that the three rate constants are now on the same side of theequation. As the name implies, these terms are constants, so we can actuallycombine them into one term. This new constant is termed the Michaelisconstant and is written KM.

. . . . (6)

Notice that the three rate constants in the definition of KM are actuallyinverted (the other way up) compared with our previous equation. This is a‘trick’ that makes for easier calculation at a later stage. Substituting thisdefinition of KM into our previous equation now gives us:

. . . . (7)

The total amount of enzyme in the system must be the same throughoutthe experiment, but it can either be free (unbound) E or in complex with

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substrate, ES. If we term the total enzyme E0, this relationship can be writtenout:

. . . (8)

This can be rearranged (by subtracting [ES] from each side) to give: . . . . (9)

So, the [E] free in solution is equal to the total amount of enzyme minusthe amount that has substrate bound. Substituting this definition of [E] backinto equation 2 gives us:

. . . (10)

This can now be rearranged in several steps. First of all, open the bracketso that the terms [E0] and [ES] are separately multiplied by [S]

. . . (11)

Next, multiply each side by KM, this gives us: . . . (12)

Then collect the two [ES] terms together on the same side (you can eitherthink of this as adding [ES][S] to both sides or as ‘carry over and change thesign’ – your preference will probably be an indication of how long ago you wentto school). This gives:

. . . (13)

Then because both terms on the right-hand side are multiplied by [ES]we can collect them together into a bracket:

. . . (14)

Dividing both sides by (KM + [S]) now gives us:

. . . (15)

Substituting this left-hand side into in place of [ES] results in:

. . . . (16)

The maximum rate, which we can call Vmax, would be achieved when allof the enzyme molecules have substrate bound. Under conditions when [S] ismuch greater than [E], it is fair to assume that all E will be in the form ES. Therefore [E0] = [ES]. Thinking again about Equation 1, we could substitutethe term Vmax for v and [E0] for [ES]. This would give us:

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. . . (17)

Notice that k2[E0] was present in our previous equation, so we can replacethis with Vmax, giving a final equation:

. . . (18)

This final equation is actually called the Michaelis-Menten equation.

Perhaps this derivation still leaves you puzzled about the importance ofthe Michaelis-Menten equation. The significance becomes clearer when youconsider the case when the rate of reaction (v) is exactly half of the maximalreaction rate (Vmax). Under those circumstances, the Michaelis-Mentenequation could be written:

. . . (19)

On dividing both sides by Vmax this becomes:

. . . (20)

Multiplying both sides by (KM + [S]) gives:

. . . (21)

And then multiplying both sides by 2 further resolves the equation to: . . . (22)

2[S] on the right-hand side is the same as [S] + [S], so we can take awayone [S] from each side. Thus when the rate of the reaction is half of themaximum rate:

. . . (23)

The KM of an enzyme is therefore the substrate concentration at whichthe reaction occurs at half of the maximum rate.

Fig. 41: Graph showing the Km status of an enzyme

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12.2 ENZYME INHIBITORSReversible inhibitors bind to enzymes with non-covalent interactions such

as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weakbonds between the inhibitor and the active site combine to produce strongand specific binding. In contrast to substrates and irreversible inhibitors,reversible inhibitors generally do not undergo chemical reactions when boundto the enzyme and can be easily removed by dilution or dialysis.There arethree kinds of reversible enzyme inhibitors. They are classified according tothe effect of varying the concentration of the enzyme’s substrate on theinhibitor.

A competitive inhibition, where the substrate and inhibitor cannot bind tothe enzyme at the same time. This usually results from the inhibitor havingan affinity for the active site of an enzyme where the substrate also binds;the substrate and inhibitor compete for access to the enzyme’s active site.This type of inhibition can be overcome by sufficiently high concentrations ofsubstrate, i.e., by out-competing the inhibitor. Competitive inhibitors are oftensimilar in structure to the real substrate.

Mixed inhibition, where the inhibitor can bind to the enzyme at the sametime as the enzyme’s substrate. However, the binding of the inhibitor affectsthe binding of the substrate, and vice versa. This type of inhibition can bereduced, but not overcome by increasing concentrations of substrate. Althoughit is possible for mixed-type inhibitors to bind in the active site, this type ofinhibition generally results from an allosteric effect where the inhibitor bindsto a different site on an enzyme. Inhibitor binding to this allosteric site changesthe conformation (i.e., tertiary structure or three-dimensional shape) of theenzyme so that the affinity of the substrate for the active site is reduced.

Non-competitive inhibition is a form of mixed inhibition where the bindingof the inhibitor to the enzyme reduces its activity but does not affect thebinding of substrate. As a result, the extent of inhibition depends only on theconcentration of the inhibitor.

12.3 ENZYME ACTIVATOR:A substance, other than the catalyst or one of the substrates, that increases

the rate of a catalysed reaction without itself being consumed; the process iscalled activation. An activator of an enzyme-catalysed reaction may be calledenzyme activator, if it acts by binding to the enzyme eg., fructose 2,6-bisphosphate, which activates phosphofructokinase 1 and increases the rateof glycolysis in response to the hormone glucagon.

12.4 LET US SUM UPMichaelis-Menten kinetics describes the kinetics of many enzymes.

To determine the maximum rate of an enzyme mediated reaction, the

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substrate concentration ([S]) is increased until a constant rate of productformation is achieved.

This is the maximum velocity (Vmax) of the enzyme. This equation can bederived from the basic substate-product enzyme reaction.

Enzyme inhibitors are molecules that bind to enzymes and decrease theiractivity. The binding of an inhibitor can stop a substrate from entering theenzyme’s active site and/or hinder the enzyme from catalysing its reaction.

Many drug molecules are enzyme inhibitors, so their discovery andimprovement is an active area of research in biochemistry and pharmacology.

Reversible inhibitors bind to enzymes with non-covalent interactions suchas hydrogen bonds, hydrophobic interactions and ionic bonds.

12.5 POINTS FOR DISCUSSIONDo a critical analysis of Michaelis – Menton kinetics and the equations

involved.

12.6 CHECK YOUR PROGRESSDescribe about enzyme inhibitors and enzyme activators?Note: a) Please don’t proceed till you attempt the above question.


12.7 LESSON –END ACTIVITIESExplain the Michaelis menton equation.What are enzyme inhibitors? Explain the types of enzyme inhibitors.

12.8 REFERENCES





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LESSON - 13

MECHANISM OF ENZYME ACTION

CONTENTS


13.1 Lack and Key model of the active site

13.2 Induced fit model of the active site

13.3 Let us Sum Up




13.7 References

13.0 AIMS AND OBJECTIVESTo learn the types of the active site and their significance.

13.1 LOCK AND KEY MODEL OF THE ACTIVE SITE:In the picture below, the active site of the enzyme and the substrate have

complementary shapes. This is so illustrated to indicate that the enzyme canrecognize the substrate based, at least in part, on its shape. Secondly, it ismeant to illustrate that the enzyme and substrate form a very closeinteraction. Thirdly, it shows that the substrate will preferentially bind tothe active site and not to other sites on the enzyme.

Fig. 42 : Lack and key model

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13.2 INDUCED FIT MODEL OF THE ACTIVE SITE:In the induced fit model of enzyme-substrate binding, the shape of the

active site of the unbound enzyme is not the exact complement of the shapeof the substrate. However, the enzyme does bind to the substrate. After bindingof the enzyme to the substrate is initiated, a conformational change in theshape of the active site which results in a new shape of the active site that iscomplementary to the shape of the substrate.

Fig. 43 : Induced fit model of the active site

ISOZYMESIsozymes were first described by Hunter and Markert (1957) who defined

them as different variants of the same enzyme having identical functions andpresent in the same individual. This definition encompasses (1) enzyme variantsthat are the product of different genes and thus represent different loci(described as isozymes) and (2) enzymes that are the product of differentalleles of the same gene (described as allozymes).

Isozymes are usually the result of gene duplication, but can also arisefrom polyploidisation or nucleic acid hybridization. Over evolutionary time, ifthe function of the new variant remains identical to the original, then it islikely that one or the other will be lost as mutations accumulate, resulting ina pseudogene. However, if the mutations do not immediately prevent theenzyme from functioning, but instead modify either its function, or its patternof gene expression, then the two variants may both be favoured by naturalselection and become specialised to different functions. For example, theymay be expressed at different stages of development or in different tissues.Glucokinase, a variant of hexokinase which is not inhibited by glucose 6-phosphate. Its different regulatory features and lower affinity for glucose(compared to other hexokinases), allows it to serve different functions incells of specific organs, such as control of insulin release by the beta cells ofthe pancreas, or initiation of glycogen synthesis by liver cells. Both of theseprocesses must only occur when glucose is abundant, or problems occur.

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Allozymes may result from point mutations or from insertion-deletion(indel) events that affect the DNA coding sequence of the gene. As with anyother new mutation, there are three things that may happen to a new allozyme:

It is most likely that the new allele will be non-functional in which caseit will probably result in low fitness and be removed from the population bynatural selection.

Alternatively, if the amino acid residue that is changed is in a relativelyunimportant part of the enzyme, for example a long way from the active sitethen the mutation may be selectively neutral and subject to genetic drift.

In rare cases the mutation may result in an enzyme that is more efficient,or one that can catalyse a slightly different chemical reaction, in which casethe mutation may cause an increase in fitness, and be favoured by naturalselection.

ALLOSTERIC ENZYMESEnzymes which contain regions to which small, regulatory molecules (cf.

effector) may bind in addition to and separate from substrate binding sites.On binding the effector, the catalytic activity of the enzyme towards thesubstrate may be enhanced, in which case the effector is an activator, orreduced, in which case it is an inhibitor. In addition to simple enzymes thatinteract only with substrates and inhibitors,there is a class of enzymes thatbind substrate and small, physiologically important molecules in ways otherthan those described above. These are known as allosteric enzymes; thesmall regulatory molecules to which they bind are known as effectors. If youexamine the Michaelis-Menten equation you will find that an increase in Vfrom 0.1 to 0.9 Vmax requires an 81-fold change in substrate concentration.In other words the velocity is rather insensitive to substrate concentration.Allosteric enzymes are “co-operative” systems,in which a small change inone parameter, e.g. substrate, inhibitor, activator concentration, brings abouta large change in velocity. A consequence of a cooperative system is that theV vs. S plot is no longer hyperbolic.To understand allosterism one mustunderstand that it is based on ligand interactions and conformational changes.

Allosteric effectors bring about catalytic modification by binding to theenzyme at distinct allosteric sites, well removed from the catalytic site, andcausing conformational changes that are transmitted through the bulk of theprotein to the catalytically active site(s). The hallmark of effectors is thatwhen they bind to enzymes, they alter the catalytic properties of an enzyme’sactive site. Those that increase catalytic activity are known as positiveeffectors. Effectors that reduce or inhibit catalytic activity are negativeeffectors.

Most allosteric enzymes are oligomeric (consisting of multiple subunits);generally they are located at or near branch points in metabolic pathways,where they are influential in directing substrates along one or another of the

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available metabolic paths. The effectors that modulate the activity of theseallosteric enzymes are of two types. Those activating and inhibiting effectorsthat bind at allosteric sites are called heterotropic effectors. These effectorscan assume a vast diversity of chemical forms, ranging from simple inorganicmolecules to complex nucleotides such as cyclic adenosine monophosphate(cAMP). Their single defining feature is that they are not identical to thesubstrate.The substrate itself induces distant allosteric effects when it bindsto the catalytic site. Substrates acting as effectors are said to be homotropiceffectors. When the substrate is the effector, it can act as such, either bybinding to the substrate-binding site, or to an allosteric effector site. Whenthe substrate binds to the catalytic site it transmits an activity-modulatingeffect to other subunits of the molecule. Often used as the model of ahomotropic effector is haemoglobin, although it is not a branch-point enzymeand thus does not fit the definition on all counts.

13.3 LET US SUM UPCoenzymes are small organic non-protein molecules that carry chemical

groups between enzymes. Examples of these are ATP (adenosine triphosphate)which is changed to ADP (adenosine diphosphate) in many reactions and NAD(nicotinamide adenine dinucleotide) which is changed to NADH (the reducedform of NAD).

A cofactor is a non-protein chemical compound that is bound tightly to anenzyme and is required for catalysis.

Isozymes are usually the result of gene duplication, but can also arisefrom polyploidisation or nucleic acid hybridization.

They are different variants of the same enzyme having identical functionsand present in the same individual.

Allozymes may result from point mutations or from insertion-deletion(indel) events that affect the DNA coding sequence of the gene.

Allosteric enzymes are “co-operative” systems,in which a small change inone parameter.

13.4 POINTS FOR DISCUSSIONDo a comparative study on isozymes and allozymes.

13.5 CHECK YOUR PROGRESSExplain Isoenzymes and allosteric enzyme?Note: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer

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13.6 LESSON –END ACTIVITIES

Explain coenzymes and metal cofactors?Describe isoenzymes with example.Explain allosteric enzymes in detail.How enzymes are used as molecular markers?

13.7 REFERENCES





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UNIT V

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LESSON - 14

CELL CYCLE

CONTENTS


14.1 Mitosis

14.2 Phases of Mitosis

14.2.1 Metaphase

14.2.2 Anaphase

14.2.3 Telophase

14.2.4 Cytokinesis

14.2.5 G1 phase

14.2.6 S phase

14.2.7 G2 phase

14.2.8 G0 phase

14.2.9 Regulation of cell cycle

14.2.10 Role of Cyclins and CDKs

14.2.11 General mechanism of cyclin-CDK interaction

14.2.12 Specific action of cyclin-CDK complexes

14.2.13 Cell cycle inhibitors

14.3 Let us Sum Up



14.6 Lesson-End Activities

14.7 References

14.0 AIMS AND OBJECTIVESTo know the stages of Mitosis elaborately.

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The cell cycle, or cell-division cycle, is the series of events that takeplace in a eukaryotic cell leading to its replication. These events can bedivided in two broad periods: interphase—during which the cell grows,accumulating nutrients needed for mitosis and duplicating its DNA—and themitotic (M) phase, during which the cell splits itself into two distinct cells,often called “daughter cells”. The cell-division cycle is an essential processby which a single-celled fertilized egg develops into a mature organism, aswell as the process by which hair, skin, blood cells, and some internal organsare renewed.The cell cycle consists of four distinct phases: G1 phase, S phase,G2 phase (collectively known as interphase) and M phase. M phase is itselfcomposed of two tightly coupled processes: mitosis, in which the cell’schromosomes are divided between the two daughter cells, and cytokinesis, inwhich the cell’s cytoplasm divides forming distinct cells. Activation of eachphase is dependent on the proper progression and completion of the previousone. Cells that have temporarily or reversibly stopped dividing are said tohave entered a state of quiescence called G0 phase

14.1 MITOSISMitosis is the process in which a cell duplicates its chromosomes to

generate two identical cells. It is generally followed by cytokinesis whichdivides the cytoplasm and cell membrane. This results in two identical cellswith an equal distribution of organelles and other cellular components. Mitosisand cytokinesis jointly define the mitotic (M) phase of the cell cycle, thedivision of the mother cell into two sister cells, each with the genetic equivalentof the parent cell. Mitosis occurs most often in eukaryotic cells. In multicellularorganisms, the somatic cells undergo mitosis, while germ cells — cells destinedto become sperm in males or ova in females divide by a related process calledmeiosis.

Cytokinesis usually occurs in conjunction with mitosis, However, thereare many cells whose mitosis and cytokinesis occur separately, forming singlecells with multiple nuclei. This occurs most notably among the fungi andslime moulds, but is found in various different groups. Even in animals,cytokinesis and mitosis may occur independently, for instance during certainstages of fruit fly embryonic development.Errors in mitosis can either kill acell through apoptosis or cause mutations that may lead to cancer or celldeath. The process of Mitosis can be divided into seven stages: Preprophase,Prophase, Prometaphase, Metaphase, Anaphase, Telophase and Cytokinesis.

Interphase: The mitotic phase is a relatively short period of the cell cycle.It alternates with the much longer interphase, where the cell prepares itselffor cell division. Interphase is divided into three phases, G1 (first gap), S(synthesis), and G2 (second gap). During all three phases, the cell grows byproducing proteins and cytoplasmic organelles. However, chromosomes arereplicated only during the S phase. Thus, a cell grows (G1), grows as itduplicates its chromosomes (S), grows more and prepares for mitosis (G2),and divides (M).

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After M phase, the daughter cells each begin interphase of a new cycle.Although the various stages of interphase are not usually morphologicallydistinguishable, each phase of the cell cycle has a distinct set of specializedbiochemical processes that prepare the cell for initiation of cell division.

Preprophase: In plant cells only, prophase is preceded by a pre-prophasestage and followed by a post-prophase stage. In plant cells that are highlyvacuolated and somewhat amorphoric, the nucleus has to migrate into thecenter of the cell before mitosis can begin. This is achieved through theformation of a phragmosome, a transverse sheet of cytoplasm that bisects thecell along the future plane of cell division. In addition to phragmosomeformation, preprophase is characterized by the formation of a ring ofmicrotubules and actin filaments (called preprophase band) underneath theplasmamembrane around the equatorial plane of the future mitotic spindleand predicting the position of cell plate fusion during telophase. The cells ofhigher plants (such as the flowering plants) lack centrioles. Instead, spindlemicrotubules aggregate on the surface of the nuclear envelope duringprophase. The preprophase band disappears during nuclear envelopedisassembly and spindle formation in prometaphase.

Prophase: Normally, the genetic material in the nucleus is in a looselybundled coil called chromatin. At the onset of prophase, chromatin condensestogether into a highly ordered structure called a chromosome. Since thegenetic material has already been duplicated earlier in S phase, the replicatedchromosomes have two sister chromatids, bound together at the centromereby the cohesion complex. Chromosomes are visible at high magnificationthrough a light microscope. Close to the nucleus are two centrosomes. Eachcentrosome, which was replicated earlier independent of mitosis, acts as acoordinating center for the cell’s microtubules. The two centrosomes nucleatemicrotubules (or microfibrils) (which may be thought of as cellular ropes) bypolymerizing soluble tubulin present in the cytoplasm. Molecular motor proteinscreate repulsive forces that will push the centrosomes to opposite side of thenucleus. The centrosomes are only present in animals. In plants themicrotubules form independently. Some centrosomials contain a pair ofcentrioles that may help organize microtubule assembly, but they are notessential to formation of the mitotic spindle.

Prometaphase: The nuclear envelope disassembles and microtubulesinvade the nuclear space. This is called open mitosis, and it occurs in mostmulticellular organisms. Fungi and some protists, such as algae ortrichomonads, undergo a variation called closed mitosis where the spindleforms inside the nucleus or its microtubules are able to penetrate an intactnuclear envelope. Each chromosome forms two kinetochores at thecentromere, one attached at each chromatid. A kinetochore is a complexprotein structure that is analogous to a ring for the microtubule hook; it isthe point where microtubules attach themselves to the chromosome. Althoughthe kinetochore structure and function are not fully understood, it is known

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that it contains some form of molecular motor. When a microtubule connectswith the kinetochore, the motor activates, using energy from ATP to “crawl”up the tube toward the originating centrosome. This motor activity, coupledwith polymerisation and depolymerisation of microtubules, provides the pullingforce necessary to later separate the chromosome’s two chromatids.

Fig. 44 : Mitosis - Phases

When the spindle grows to sufficient length, usually at least 7 nanometers,kinetochore microtubules begin searching for kinetochores to attach to. Anumber of nonkinetochore microtubules find and interact with correspondingnonkinetochore microtubules from the opposite centrosome to form the mitoticspindle. Prometaphase is sometimes considered part of prophase.

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14.2 PHASES OF MITOSIS

14.2.1 METAPHASE:As microtubules find and attach to kinetochores in prometaphase, the

centromeres of the chromosomes convene along the metaphase plate orequatorial plane, an imaginary line that is equidistant from the two centrosomepoles.[9] This even alignment is due to the counterbalance of the pullingpowers generated by the opposing kinetochores, analogous to a tug-of-warbetween equally strong people. In certain types of cells, chromosomes do notline up at the metaphase plate and instead move back and forth between thepoles randomly, only roughly lining up along the midline. Metaphase comesfrom the Greek word for “metanosis” ìåôá meaning “after.” Because properchromosome separation requires that every kinetochore be attached to abundle of microtubules (spindle fibers) , it is thought that unattachedkinetochores generate a signal to prevent premature progression toanaphasewithout all chromosomes being aligned. The signal creates themitotic spindle checkpoint.

14.2.2. ANAPHASEWhen every kinetochore is attached to a cluster of microtubules and the

chromosomes have lined up along the metaphase plate, the cell proceeds toanaphase (from the Greek áíá meaning “up,” “against,” “back,” or “re-”).

Two events then occur; first, the proteins that bind sister chromatidstogether are cleaved, allowing them to separate. These sister chromatids arehereafter independent sister chromosomes. They are pulled apart byshortening kinetochore microtubules and toward the respective centrosomesto which they are attached. This is followed by the elongation of thenonkinetochore microtubules, which pushes the centrosomes (and the set ofchromosomes to which they are attached) apart to opposite ends of the cell.These three stages are sometimes called early, mid and late anaphase. Earlyanaphase is usually defined as the separation of the sister chromatids. Midanaphase occurs with the reunification of certain metastic chromatids. Lateanaphase is the elongation of the microtubules and the microtubules beingpulled further apart. At the end of anaphase, the cell has succeeded inseparating identical copies of the genetic material into two distinct populations.

14.2.3. TELOPHASE:Telophase (from the Greek ôåëïò meaning “end”) is a reversal of prophase

and prometaphase events. It “cleans up” the after effects of mitosis. Attelophase, the nonkinetochore microtubules continue to lengthen, elongatingthe cell even more. Corresponding sister chromosomes attach at oppositeends of the cell. A new nuclear envelope, using fragments of the parent cell’snuclear membrane, forms around each set of separated sister chromosomes.Both sets of chromosomes, now surrounded by new nuclei, unfold back into

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chromatin. Mitosis is complete, but cell division is not yet complete.

14.2.4. CYTOKINESISCytokinesis is often mistakenly thought to be the final part of telophase;

however cytokinesis is a separate process that begins after telophase.Cytokinesis is technically not even a phase of mitosis, but rather a separateprocess, necessary for completing cell division. In animal cells, a cleavagefurrow (pinch) containing a contractile ring develops where the metaphaseplate used to be, pinching off the separated nuclei. In both animal and plantcells, cell division is also driven by vesicles derived from the Golgi apparatus,which move along microtubules to the middle of the cell. In plants this structurecoalesces into a cell plate at the center of the phragmoplast and develops intoa cell wall, separating the two nuclei. The phragmoplast is a microtubulestructure typical for higher plants, whereas some green algae use a phycoplastmicrotubule array during cytokinesis. Each daughter cell has a completecopy of the genome of its parent cell. The end of cytokinesis marks the end ofthe M-phase.

14.2.5. G1 PHASEThe first phase within interphase, from the end of the previous M phase

till the beginning of DNA synthesis is called G1 (G indicating gap or growth).During this phase the biosynthetic activities of the cell, which had beenconsiderably slowed down during M phase, resume at a high rate. This phaseis marked by synthesis of various enzymes that are required in S phase,mainly those needed for DNA replication. Duration of G1 is highly variable,even among different cells of the same species.

14.2.6. S PHASEThe ensuing S phase starts when DNA synthesis commences; when it is

complete, all of the chromosomes have been replicated, i.e., each chromosomehas two (sister) chromatids. Thus, during this phase, the amount of DNA inthe cell has effectively doubled, though the ploidy of the cell remains thesame. Rates of RNA transcription and protein synthesis are very low duringthis phase. An exception to this is histone production, most of which occursduring the S phase. The duration of S phase is relatively constant amongcells of the same species.

14.2.7. G2 PHASEThe cell then enters the G2 phase, which lasts until the cell enters the

next round of mitosis. Again, significant protein synthesis occurs during thisphase, mainly involving the production of microtubules, which are requiredduring the process of mitosis. Inhibition of protein synthesis during G2 phaseprevents the cell from undergoing mitosis.

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14.2.8. G0 PHASEThe term “post-mitotic” is sometimes used to refer to both quiescent and

senescent cells. Nonproliferative cells in multicellular eukaryotes generallyenter the quiescent G0 state from G1 and may remain quiescent for longperiods of time, possibly indefinitely (as is often the case for neurons). This isvery common for cells that are fully differentiated. Cellular senescence is astate that occurs in response to DNA damage or degradation that would makea cell’s progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis. Some cell types in matureorganisms, such as parenchymal cells of the liver and kidney, enter the G0phase semi-permanently and can only be induced to begin dividing againunder very specific circumstances; other types, such as epithelial cells,continue to divide throughout an organism’s life.

14.2.9. REGULATION OF CELL CYCLERegulation of the cell cycle involves steps crucial to the cell, including

detecting and repairing genetic damage, and provision of various checks toprevent uncontrolled cell division. The molecular events that control the cellcycle are ordered and directional; that is, each process occurs in a sequentialfashion and it is impossible to “reverse” the cycle.

14.2.10. ROLE OF CYCLINS AND CDKSTwo key classes of regulatory molecules, cyclins and cyclin-dependent

kinases (CDKs), determine a cell’s progress through the cell cycle. Leland H.Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize inPhysiology or Medicine for their discovery of these central molecules. Many ofthe genes encoding cyclins and CDKs are conserved among all eukaryotes,but in general more complex organisms have more elaborate cell cycle controlsystems that incorporate more individual components. Many of the relevantgenes were first identified by studying yeast, especially Saccharomycescerevisiae; genetic nomenclature in yeast dubs many of these genes cdc (for“cell division cycle”) followed by an identifying number, e.g., cdc25.

Cyclins form the regulatory subunits and CDKs the catalytic subunits ofan activated heterodimer; cyclins have no catalytic activity and CDKs areinactive in the absence of a partner cyclin. When activated by a bound cyclin,CDKs perform a common biochemical reaction called phosphorylation thatactivates or inactivates target proteins to orchestrate coordinated entry intothe next phase of the cell cycle. Different cyclin-CDK combinations determinethe downstream proteins targeted. CDKs are constitutively expressed in cellswhereas cyclins are synthesised at specific stages of the cell cycle, in responseto various molecular signals.

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14.2.11. GENERAL MECHANISM OF CYCLIN-CDK INTERACTIONUpon receiving a pro-mitotic extracellular signal, G1 cyclin-CDK complexes

become active to prepare the cell for S phase, promoting the expression oftranscription factors that in turn promote the expression of S cyclins and ofenzymes required for DNA replication. The G1 cyclin-CDK complexes alsopromote the degradation of molecules that function as S phase inhibitors bytargeting them for ubiquitination. Once a protein has been ubiquitinated, itis targeted for proteolytic degradation by the proteasome. Active S cyclin-CDKcomplexes phosphorylate proteins that make up the pre-replication complexesassembled during G1 phase on DNA replication origins. The phosphorylationserves two purposes: to activate each already-assembled pre-replicationcomplex, and to prevent new complexes from forming. This ensures that everyportion of the cell’s genome will be replicated once and only once. The reasonfor prevention of gaps in replication is fairly clear, because daughter cellsthat are missing all or part of crucial genes will die. However, for reasonsrelated to gene copy number effects, possession of extra copies of certaingenes would also prove deleterious to the daughter cells.

Mitotic cyclin-CDK complexes, which are synthesized but inactivatedduring S and G2 phases, promote the initiation of mitosis by stimulatingdownstream proteins involved in chromosome condensation and mitotic spindleassembly. A critical complex activated during this process is a ubiquitin ligaseknown as the anaphase-promoting complex (APC), which promotes degradationof structural proteins associated with the chromosomal kinetochore. APC alsotargets the mitotic cyclins for degradation, ensuring that telophase andcytokinesis can proceed.

14.2.12. SPECIFIC ACTION OF CYCLIN-CDK COMPLEXESCyclin D is the first cyclin produced in the cell cycle, in response to

extracellular signals (eg. growth factors). Cyclin D binds to existing CDK4,forming the active cyclin D-CDK4 complex. Cyclin D-CDK4 complex in turnphosphorylates the retinoblastoma susceptibility protein (RB). Thehyperphosphorylated RB dissociates from the E2F/DP1/RB complex (whichwas bound to the E2F responsive genes, effectively “blocking” them fromtranscription), activating E2F. Activation of E2F results in transcription ofvarious genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc.Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex,which pushes the cell from G1 to S phase (G1/S transition). Cyclin A alongwith CDK2 forms the cyclin A-CDK2 complex, which initiates the G2/Mtransition. Cyclin B-CDK1 complex activation causes breakdown of nuclearenvelope and initiation of prophase, and subsequently, it’s deactivation causesthe cell to exit mitosis.

14.2.13. CELL CYCLE INHIBITORSTwo families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of

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Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle.Because these genes are instrumental in prevention of tumor formation,they are known as tumor suppressors.The cip/kip family includes the genesp21, p27 and p57. They halt cell cycle in G1 phase, by binding to, andinactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, istriggered by DNA damage eg. due to radiation). p27 is activated by TransformingGrowth Factor â (TGF â), a growth inhibitor.The INK4a/ARF family includesp16INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, andp14arf which prevents p53 degradation.

14.3 LET US SUM UPThe cell cycle, or cell-division cycle, is the series of events that take

place in a eukaryotic cell leading to its replication.

Mitosis is the process in which a cell duplicates its chromosomes togenerate two identical cells.

Mitosis occurs most often in eukaryotic cells.

Mitosis contains Interphase, pre-prophase, prophase, Prometaphase,metaphase, Anaphase, Telophase, and Cytokinesis.

Interphase is divided into three phases, G1 (first gap), S (synthesis), andG2 (second gap).

Regulation of the cell cycle involves detecting and repairing geneticdamage, and provision of various checks to prevent uncontrolled cell division.

Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor ofKinase 4/Alternative Reading Frame) prevent the progression of the cell cycle.

Because these genes are instrumental in prevention of tumor formation,they are known as tumor suppressors.

14.4 POINTS FOR DISCUSSION“Mitosis is an essential part of the regulations of cell cycle” – Express

your views.

14.5 CHECK YOUR PROGRESSExplain the prophase and prometaphase of mitosis.Note: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer..............................................................................................................................................................................................................................................................................................................................................

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14.6 LESSON-END ACTIVITIESDefine cell cycleWhat does it mean by the term “interphase” in cell division?Describe the different steps involved in mitosisWrite notes on the regulation of cell cycle

14.7 REFERENCES





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LESSON - 15

MEIOSIS

CONTENTS


15.1 Meiosis – I

15.1.1 Prophase – I

15.2 Meiosis – II

15.3 Let us Sum Up




15.7 References

15.0 AIMS AND OBJECTIVESTo learn and understand the stages of meisosi.

In biology, meiosis is the process by which one diploid eukaryotic celldivides to generate four haploid cells often called gametes. The word “meiosis”comes from the Greek meioun, meaning “to make smaller,” since it results ina reduction in chromosome number in the gamete cell. During meiosis, thegenome of a diploid germ cell, which is composed of long segments of DNApackaged into chromosomes, undergoes DNA replication followed by two roundsof division, resulting in haploid cells called gametes. Each gamete containsone complete set of chromosomes, or half of the genetic content of the originalcell. These resultant haploid cells can fuse with other haploid cells of theopposite sex or mating type during fertilization to create a new diploid cell, orzygote. Thus, the division mechanism of meiosis is a reciprocal process to thejoining of two genomes that occurs at fertilization. Because the chromosomesof each parent undergo genetic recombination during meiosis, each gamete,and thus each zygote, will have a unique genetic blueprint encoded in itsDNA. In other words, meiosis and sexual reproduction produce geneticvariation.

Growth 1 (G1) phase: Immediately follows cytogenesis. This is a veryactive period, where cell synthesizes its vast array of proteins, including theenzymes and structural proteins it will need for growth. In G1 stage each ofthe 46 human chromosomes consists of a single (very long) molecule of DNA

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Synthesis (S) phase: The genetic material is replicated: each of itschromosomes duplicates. The cell is still diploid, however, because it stillcontains the same number of centromeres. However, the identical sisterchromatids are in the chromatin form because spiralisation and condensationinto denser chromosomes have not taken place yet. It will take place inprophase I in meiosis.

Growth 2 (G2) phase: The cell continues to grow making the cell larger.Interphase is immediately followed by meiosis I and meiosis II. Meiosis Iconsists of segregating the homologous chromosomes from each other, thendividing the diploid cell into two haploid cells each containing one of thesegregates. Meiosis II consists of decoupling each chromosome’s sister strands(chromatids), segregating the DNA into two sets of strands (each set containingone of each homolog), and dividing both haploid, duplicated cells to producefour haploid, unduplicated cells. Meiosis I and II are both divided into prophase,metaphase, anaphase, and telophase subphases, similar in purpose to theiranalogous subphases in the mitotic cell cycle. Therefore, meiosis encompassesthe interphase (G1, S, G2), meiosis I (prophase I, metaphase I, anaphase I,telophase I), and meiosis II (prophase II, metaphase II, anaphase II, telophaseII).

15.1 MEIOSIS I

15.1.1 PROPHASE ILeptotene: The first stage of prophase I is the leptotene stage, also known

as leptonema, from Greek words meaning “thin threads.” During this stage,individual chromosomes begin to condense into long strands within thenucleus. However the two sister chromatids are still so tightly bound thatthey are indistinguishable from one another.

Zygotene: The zygotene stage, also known as zygonema, from Greek wordsmeaning “paired threads,” occurs as the chromosomes approximately line upwith each other into homologous chromosomes. The combined homologouschromosomes are said to be bivalent. They may also be referred to as a tetrad,a reference to the four sister chromatids. The two chromatids become “zipped”together, forming the synaptonemal complex, in a process known as synapsis.

Pachytene: The pachytene stage, also known as pachynema, from Greekwords meaning “thick threads, contains the chromosomal crossover. Nonsisterchromatids of homologous chromosomes randomly exchange segments ofgenetic information over regions of homology. (Sex chromosomes, however,are not identical, and only exchange information over a small region ofhomology.) Exchange takes place at sites where recombination nodules haveformed. The exchange of information between the non-sister chromatidsresults in a recombination of information; each chromosome has the completeset of information it had before, and there are no gaps formed as a result ofthe process. Because the chromosomes cannot be distinguished in the

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synaptonemal complex, the actual act of crossing over is not perceivablethrough the microscope.

Diplotene: During the diplotene stage, also known as diplonema, fromGreek words meaning “two threads,” the synaptonemal complex degradesand homologous chromosomes separate from one another a little. Thechromosomes themselves uncoil a bit, allowing some transcription of DNA.However, the homologous chromosomes of each bivalent remain tightly boundat chiasmata, the regions where crossing over occurred.

Diakinesis: Chromosomes condense further during the diakinesis stage,from Greek words meaning “moving through.”[1] This is the first point inmeiosis where the four parts of the tetrads are actually visible. Sites of crossingover entangle together, effectively overlapping, making chiasmata clearlyvisible. Other than this observation, the rest of the stage closely resemblesprometaphase of mitosis; the nucleoli disappears, the nuclear membranedisintegrates into vesicles, and the meiotic spindle begins to form.

SYNCHRONOUS PROCESSESDuring these stages, centrioles are migrating to the two poles of the cell.

These centrioles, which were duplicated during interphase, function asmicrotubule coordinating centers. Centrioles sprout microtubules, essentiallycellular ropes and poles, during crossing over. They invade the nuclearmembrane after it disintegrates, attaching to the chromosomes at thekinetochore. The kinetochore functions as a motor, pulling the chromosomealong the attached microtubule toward the originating centriole, like a trainon a track. There are two kinetochores on each tetrad, one for each centrosome.

Prophase I is the longest phase in meiosis.Microtubules that attach tothe kinetochores are known as kinetochore microtubules. Other microtubuleswill interact with microtubules from the opposite centriole. These are callednonkinetochore microtubules.

Metaphase I: Homologous pairs move together along the phase plate: askinetochore microtubules from both centrioles attach to their respectivekinetochores, the homologous chromosomes align along an equatorial planethat bisects the spindle, due to continuous counterbalancing forces exertedon the bivalents by the microtubules emanating from the two kinetochores.The physical basis of the independent assortment of chromosomes is therandom orientation of each bivalent along the metaphase plate.

Anaphase I: Kinetochore microtubules shorten, severing the recombinationnodules and pulling homologous chromosomes apart. Since each chromosomeonly has one kinetochore, whole chromosomes are pulled toward opposingpoles, forming two diploid sets. Each chromosome still contains a pair of sisterchromatids. Nonkinetochore microtubules lengthen, pushing the centriolesfurther apart. The cell elongates in preparation for division down the middle.In prophase 1 the DNA coils tightly and individual chromosomes become visible

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under the light microscope. Homologous chromosomes closely associated insynapsis and they exchange segments by crossing over.

Telophase I: The first meiotic division effectively ends when thecentromeres arrive at the poles. Each daughter cell now has half the numberof chromosomes but each chromosome consists of a pair of chromatids. Thiseffect produces a variety of responses from the neuro-synrchromatic enzyme,also known as NSE. The microtubules that make up the spindle networkdisappear, and a new nuclear membrane surrounds each haploid set. Thechromosomes uncoil back into chromatin. Cytokinesis, the pinching of thecell membrane in animal cells or the formation of the cell wall in plant cells,occurs, completing the creation of two daughter cells. Cells enter a period ofrest known as interkinesis or interphase II. No DNA replication occurs duringthis stage. Note that many plants skip telophase I and interphase II, goingimmediately into prophase II.

15.2 MEIOSIS IIProphase II takes an inversely proportional time compared to telophase I.

In this prophase we see the disappearance of the nucleoli and the nuclearenvelope again as well as the shortening and thickening of the chromatids.Centrioles move to the polar regions and are arranged by spindle fibres. Thenew equatorial plane is rotated by 90 degrees when compared to meiosis I,perpendicular to the previous plane.

In metaphase II, the centromeres contain three kinetochores, organizingfibers from the centrosomes on each side. This is followed by anaphase II,where the centromeres are cleaved, allowing the kinetochores to pull thesister chromatids apart. The sister chromatids by convention are now calledsister chromosomes, and they are pulled toward opposing poles. The processends with telophase II, which is similar to telophase I, marked by uncoiling,lengthening, and disappearance of the chromosomes occur as thedisappearance of the microtubules. Nuclear envelopes reform; cleavage orcell wall formation eventually produces a total of four daughter cells, eachwith a haploid set of chromosomes. Meiosis is now complete.

SIGNIFICANCE OF MEIOSISMeiosis facilitates stable sexual reproduction without the halving of ploidy,

or chromosome count, fertilization would result in zygotes that have twicethe number of chromosomes than the zygotes from the previous generation.Successive generations would have an exponential increase in chromosomecount, resulting in an unwieldy genome that would cripple the reproductivefitness of the species. Polyploidy, the state of having three or more sets ofchromosomes, also results in developmental abnormalities or lethality.Polyploidy is poorly tolerated in animal species. Plants, however, regularlyproduce fertile, viable polyploids. Polyploidy has been implicated as an importantmechanism in plant speciation. Most importantly, however, meiosis producesgenetic variety in gametes that propagate to offspring. Recombination and

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independent assortment allow for a greater diversity of genotypes in thepopulation. As a system of creating diversity, meiosis allows a species tomaintain stability under environmental changes.

15.3 LET US SUM UPMeiosis is the process by which one diploid eukaryotic cell divides to

generate four haploid cells often called gametes.

Meiosis encompasses the interphase (G1, S, G2), meiosis I (prophase I,metaphase I, anaphase I, telophase I), and meiosis II (prophase II, metaphaseII, anaphase II, telophase II).

Meiosis facilitates stable sexual reproduction.

The normal separation of chromosomes in Meiosis I or sister chromatidsin meiosis II is termed disjunction.

15.4 POINTS FOR DISCUSSIONDo a comparative study between mitosis and meiosis.

15.5 CHECK YOUR PROGRESSGive an account on meiosis I and the steps involved in it.Note: a) Please don’t proceed till you attempt the above question.


15.6 LESSON-END ACTIVITIESDefine meiosisWhat is Diakinesis?Write the mechanism of meiosisWhat is the significance of meiosis in organisms?What are Down’s syndrome, Patau syndrome, Edward syndrome,

Klinefelter syndrome, Turner syndrome?

15.7 REFERENCES



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LESSON - 16

DNA REPLICATION

CONTENTS


16.1 DNA structure

16.2 Lagging strand synthesis

16.3 Leading strand synthesis

16.4 Dynamics at the replication fork

16.5 Mechanism of replication

16.6 DNA replication in enbacteria initiation of replication and the

bacterial origin.

16.7 Termination of replication

16.8 Regulation of replication

16.9 Rolling circle replication

16.10 Plasmid replication

16.11 Let us Sum Up




16.15 References

16.0 AIMS AND OBJECTIVESTo study the DNA structure and its replication.

16.1 DNA STRUCTUREA DNA strand is a long polymer built from nucleotides; two complementary

DNA strands form a double helix. The two strands in the DNA backbone runanti-parallel to each other: One of the DNA strands is built in the 5' ’! 3'direction, while the complementary strand is built in the 3' ’! 5' direction (5'and 3' each mark one end of a strand). Each nucleotide consists of a phosphateand a deoxyribose sugar - forming the backbone of the DNA double helix and a

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base. When a nucleotide base forms hydrogen bonds with a complementarybase on the other strand,they form a base pair: Adenine pairs with thymineand cytosine pairs with guanine. These pairings are often expressed withC:::G and A::T where the dots indicate hydrogen bonds. A strand running inthe 5'’! 3' direction that has adenine will pair with base thymine on thecomplementary strand running in 3'’! 5' direction.

The replication fork: Many enzymes are involved in the DNA replicationfork.The replication fork is a structure which forms when DNA is beingreplicated. It is created through the action of helicase, which breaks thehydrogen bonds holding the two DNA strands together. The resulting structurehas two branching “prongs”, each one made up of a single strand of DNA.

Fig. 45 : DNA replication

16.2 LAGGING STRAND SYNTHESIS:In DNA replication, the lagging strand is the DNA strand at the opposite

side of the replication fork from the leading strand. It goes from 3' to 5' (thesenumbers indicate the position of the molecule in respect to the carbon atomsit contains). When the enzyme helicase unwinds DNA, two single strandedregions of DNA (the “replication fork”) form. DNA polymerase cannot build astrand in the 3' ’! 5' direction. Thus, the strand complementary to the 5' ’! 3'template strand (known as the lagging strand) is synthesized in short segmentsknown as Okazaki fragments. On the lagging strand, primase builds an RNAprimer in short bursts. DNA polymerase is then able to use the free 3' OHgroup on the RNA primer to synthesize DNA in the 5' ’! 3' direction. The RNAfragments are then removed (different mechanisms are used in eukaryotesand prokaryotes) and new deoxyribonucleotides are added to fill the gapswhere the RNA was present. DNA ligase is then able to ligate thedeoxyribonucleotides together, completing the synthesis of the lagging strand.

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16.3 LEADING STRAND SYNTHESIS:The leading strand is defined as the DNA strand that is synthesized in

the 5' ’! 3' direction in a continuous manner. On this strand, DNA polymeraseIII is able to synthesize DNA using the free 3'-OH group donated by a singleRNA primer (multiple RNA primers are not used) and continuous synthesisoccurs in the direction in which the replication fork is moving.

16.4 DYNAMICS AT THE REPLICATION FORK:The sliding clamp in all domains of life share a similar structure, and are

able to interact with the various processive and non-processive DNApolymerases found in cells. In addition, the sliding clamp serves as aprocessivity factor. The C-terminal end of the clamps forms loops which areable to interact with other proteins involved in DNA replication (such as DNApolymerase and the clamp loader). The inner face of the clamp allows DNA tobe threaded through it. The sliding clamp forms no specific interactions withDNA. There is a large 35A0 hole in the middle of the clamp. This allows DNAto fit through it, and water to take up the rest of the space allowing the clampto slide along the DNA. Once the polymerase reaches the end of the templateor detects double stranded DNA, the sliding clamp undergoes a conformationalchange which releases the DNA polymerase.

The clamp loader, a multisubunit protein, is able to bind to the slidingclamp and DNA polymerase. When ATP is hydrolyzed, it loses affinity for thesliding clamp allowing DNA polymerase to bind to it. Furthermore, the slidingclamp can only be bound to a polymerase as long as single stranded DNA isbeing synthesized. Once the single stranded DNA runs out, the polymerase isable to bind to the subunit on the clamp loader and move to a new position onthe lagging strand. On the leading strand, DNA polymerase III associateswith the clamp loader and is bound to the sliding clamp.Recent evidencesuggests that the enzymes and proteins involved in DNA replication remainstationary at the replication forks while DNA is looped out to maintainbidirectionality in observed in replication. This is a result of an interactionbetween DNA polymerase, the sliding clamp, and the clamp loader. DNAreplicationDNA replication differs somewhat between eukaryotic and prokaryoticcells. Much of our knowledge of the process DNA replication was derived fromthe study of E. coli, while yeast has been used as a model organism forunderstanding eukaryotic DNA replication.

MECHANISM OF REPLICATION:Once priming of DNA is complete, DNA polymerase is loaded into the DNA

and replication begins. The catalytic mechanism of DNA polymerase involvesthe use of two metal ions in the active site and a region in the active site thatcan discriminate between deoxynucleotides and ribonucleotides. The metalions are general divalent cations that help the 3'-OH to initiate a nucleophilicattack onto the alpha-phosphate of the deoxyribonucleotide and orient and

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stabilize the negatively-charged triphosphate on the deoxyribonucleotide.Nucleophillic attack by the 3'-OH on the alpha phosphate releasespyrophosphate, which is then subsequently hydrolyzed by inorganicpyrophosphatase into two phosphates. Subsequently driving DNA synthesis tocompletion.

Furthermore, DNA polymerase must be able to distinguish betweencorrectly paired bases and incorrectly paired bases. This is accomplished bydistinguishing Watson-Crick base pairs through the use of an active site pocketthat is complementary in shape to the structure of correctly paired nucleotides.This pocket has a tyrosine residue that is able to form van der Waalsinteractions with the correctly paired nucleotide. In addition, double strandedDNA in the active site has a wider and shallower minor groove that permitsthe formation of hydrogen bonds with the third nitrogen of purine bases andthe second oxygen of pyrimidine bases. Finally, the active site makes extensivehydrogen bonds with the DNA backbone. These interactions result in theDNA polymerase III closing around a correctly paired base. If a base is insertedand incorrectly paired, these interactions could not occur due to disruptionsin hydrogen bonding and van der Waals interactions. The mechanism ofreplication is similar in eukaryotes and prokaryotes.

DNA is read in the 3' ’! 5' direction, relative to the parent strand, therefore,nucleotides are synthesized (or attached to the template strand) in the 5' ’! 3'direction, relative to the daughter strand. However, one of the parent strandsof DNA is 3' ’! 5' and the other is 5' ’! 3'. To solve this, replication must proceedin opposite directions. The leading strand runs towards the replication forkand is thus synthesized in a continuous fashion, only requiring one primer.On the other hand, the lagging strand runs in the opposite direction, headingaway from the replication fork, and is synthesized in a series of short fragmentsknown as Okazaki fragments, consequently requiring many primers. The RNAprimers of Okazaki fragments are subsequently degraded by RNase H andDNA polymerase I (exonuclease), and the gap (or nick’s) are filled withdeoxyribonucleotides and sealed by the enzyme ligase.

16.6 DNA REPLICATION IN EUBACTERIA INITIATION OF REPLICATION ANDTHE BACTERIAL ORIGIN:

DNA replication in E. coli is bi-directional and originates at a single originof replication (OriC). The initiation of replication is mediated by DnaA, aprotein that binds to a region of the origin known as the DnaA box. In E. coli,there are 5 DnaA boxes, each of which contains a highly conserved 9-basepair consensus sequence 5' - TTATCCACA - 3'. Binding of DnaA to this regioncauses it to become negatively supercoiled. Following this, a region of OriCupstream of the DNA A boxes (known as DNA B boxes) melts. There are threeof these regions. Each are 13 base pairs long and rich in A-T base pairs. Thisfacilitates melting because less energy is required to break the two hydrogenbonds that form between A and T nucleotides. This region has the consensussequence 5' - GATCTNTTNTTTT - 3. Melting of the DnaB boxes requires ATP

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(which is hydrolyzed by DnaA). Following melting, DNA A recruits a hexamerichelicase (six DNA B proteins) to opposite ends of the melted DNA. This iswhere the replication fork will form. Recruitment of helicase requires sixDnaC proteins, each of which is attached to one subunit of helicase. Oncethis complex is formed, an additional five DnaA proteins bind to the originalfive DnaA proteins to form five DnaA dimers. DnaC is then released, and theprepriming complex is complete. In order for DNA replication to continue,single-strand binding proteins (SSBs) are needed to prevent the single strandsof DNA from forming any secondary structures and to prevent them fromreannealing, and DNA gyrase is needed to relieves the stress (by creatingnegative supercoils) created by the action of DNA B helicase. The unwindingof DNA by DNA B helicase allows for primase (DNA G) and RNA polymerase toprime each DNA template so that DNA synthesis can begin.

16.7 TERMINATION OF REPLICATION:Termination of DNA replication in E. coli is completed through the use of termination sequencesand the Tus protein. These sequences allow the two replication forks to pass through in only onedirection, but not the other. In order to slow down and stop the movement of the replicationfork in the termination region of the E. coli chromosome, the Tus protein is required. This proteinbinds to the termination sites, and prevents DnaB from displacing DNA strands. However, thesesequences are not required for termination of replication.

16.8 REGULATION OF REPLICATION:Regulation of DNA replication is achieved through several mechanisms.

Mechanisms of regulation involve the ratio of ATP to ADP, the ratio of DnaAprotein to DnaA boxes and the hemimethylation and sequestering of OriC.The ratio of ATP to ADP indicates that the cell has reached a specific size andis ready to divide. This “signal” occurs because in a rich medium, the cell willgrow quickly and will have a lot of excess ATP. Furthermore, DnaA bindsequally well to ATP or ADP, but only the DnaA-ATP complex is able to initiatereplication. Thus, in a fast growing cell, there will be more DnaA-ATP thanDnaA-ADP.Another mode of regulation involves the levels of DnaA in the cell.5 DnaA-DnaA dimers are needed to initiate replication. Thus, the ratio ofDnaA to the number of DnaA boxes in the cell is important. After DNAreplication is complete, this number is halved and replication cannot occuruntil the levels of DnaA protein increase.

Finally, upon completion of DNA replication, DNA is sequestered to amembrane-binding protein called SeqA. This protein binds to hemimethylatedGATC DNA sequences. This 4-base pair sequence occurs 11 times in OriC.Only the parent strand is methylated upon completion of DNA synthesis.DAM methyltransferase methylates the adenine residues in the newlysynthesized strand of DNA only if it is not bound to SeqA. The importance ofthis form of regulation is twofold: 1) OriC becomes inaccessible to DnaA and2) DnaA binds better to fully methylated DNA than hemimethylated DNA.

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16.9 ROLLING CIRCLE REPLICATION:Replication of the bacterial chromosome is known as theta (è) replication.

However, another method of replication exists in bacterial cells, known asrolling circle replication.Rolling circle replication describes a process of DNAreplication that can rapidly synthesize multiple copies of circular moleculesof DNA, such as plasmids and the genomes of bacteriophages.Rolling circlereplication is initiated by an initiator protein encoded by the plasmid orbacteriophage DNA. This protein is able to nick one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin(DSO) and remains bound to the 5'-PO4 end of the nicked strand. The free 3'-OH end is released and can serve as a primer for DNA synthesis by DNApolymerase III. Using the unnicked strand as a template, replication proceedsaround the circular DNA molecule, displacing the nicked strand as single-stranded DNA.

Continued DNA synthesis can produce multiple single-stranded linearcopies of the original DNA in a continuous head-to-tail series. These linearcopies can be converted to double-stranded circular molecules through thefollowing process: First, the initiator protein makes another nick to terminatesynthesis of the first (leading) strand. RNA polymerase and DNA polymeraseIII then replicate the single-stranded origin (SSO) DNA to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA,and DNA ligase joins the ends to make another molecule of double-strandedcircular DNA.

A striking feature of rolling circle replication is the uncoupling of thereplication of the two strands of the DNA molecule. In contrast to commonmodes of DNA replication where both the parental DNA strands are replicatedsimultaneously, in rolling circle replication one strand is replicated first (whichprotrudes after being displaced, giving the characteristic appearance) andthe second strand is replicated after completion of the first one.Rolling circlereplication has found wide uses in academic research and biotechnology, andhas been successfully used for amplification of DNA from very small amountsof starting material.

16.10 PLASMID REPLICATION:Origin and regulation:-The regulation of plasmids differs considerably

from the regulation of chromosomal replication. However, the machineryinvolved in the replication of plasmids is similar to that of chromosomalreplication. The plasmid origin is commonly termed OriV, and at this siteDNA replication is initiated. The ori region of plasmids, unlike that found onthe host chromosome, contain the genes required for its replication. In addition,the ori region determines the host range. Plasmids carrying the ColE1 originhave a narrow host range and are restricted to the relatives of E. coli. Plasmidsof utilizing the RK2 ori and ones that replicate using rolling circle replicationhave a broad host range and are compatible with gram positive and gram

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negative bacteria. Another important characteristic of the ori region is theregulation of plasmid copy number. Generally, high copy number plasmidshave mechanisms that inhibit the initiation of replication. Regulation ofplasmids based on the ColE1 origin, a high copy number origin, require anantisense RNA. A gene close to the origin, RNAII is transcribed and the 3'-OHof the transcript primes the origin only if it is cleaved by RNase H. Transcriptionof RNAI, the antisense RNA, inhibits the RNAII from priming the DNA becauseit prevents the formation of the RNA-DNA hybrid recognized by RNase H.

16.11 LET US SUM UPDNA replication is the process of copying a double-stranded DNA molecule

resulting in identical double-stranded DNA molecules.

The replication tales place in three steps. They are initiation, elongationand termination.

The lagging strand is the DNA strand at the opposite side of the replicationfork from the leading strand.

The leading strand is defined as the DNA strand that is synthesized inthe 5' ’! 3' direction in a continuous manner.

16.12 POINTS FOR DISCUSSION“DNA replication is by for the most important aspect in any organism” –

Give your opinions on this statement.

16.13 CHECK YOUR PROGRESSExplain the leading and lagging strand synthesis in DNA replicationNote: a) Please don’t proceed till you attempt the above question.

b) The space given below is for your answer.

16.14 LESSON-END ACTIVITIES1) What is leading and lagging strand synthesis2) Give short notes on steps involved in DNA replication.............................................................................................................................................................................................................................................................................................................................................................................................................................................................

16.15 REFERENCES


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LESSON - 17

DNA TRANSCRIPTION

CONTENTS


17.1 Initiation

17.2 Elongation

17.3 Termination

17.4 Prokaryotic Vs. Eukaryotic transcription

17.5 Measuring and Defecting Transcription

17.6 Transcription factories

17.7 Transcription initiation and complex

17.8 Terminology

17.9 Reverse transcription

17.10 Let us Sum Up




17.14 References

17.0 AIMS AND OBJECTIVESTo learn and understand DNA transcription.

INITIATION:In transcription, one strand of DNA, the non-coding strand, is used as a

template for RNA synthesis. As transcription proceeds in the 5' ’! 3' direction,and uses base pairing complimentarity with the DNA template to specify thecorrect copying, the DNA template strand is that oriented in the 3' ’! 5'direction. The strand that is not used as the template is called the codingstrand, and has the DNA sequence that reflects that of the RNA produced.

Transcription begins with the binding of RNA polymerase to the promoterin DNA. In prokaryotes, the RNA polymerase is a core enzyme consisting of

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five subunits: 2 á subunits, 1 â subunit, 1 â’ subunit, and 1 ù subunit. At thestart of initiation, the core enzyme is associated with a sigma factor (number70) that aids in finding the appropriate -35 and -10 basepairs downstream ofpromoter sequences. Transcription initiation is far more complex in eukaryotes,the main difference being that eukaryotic polymerases do not recognize directlytheir core promoter sequences. Unlike DNA replication, transcription doesnot need a primer to start. The DNA unwinds and produces a small opencomplex and synthesis begins on only the template strand.

17.2 ELONGATION:Unlike DNA replication, mRNA transcription can involve multiple RNA

polymerases; so many mRNA molecules can be produced from a single copy ofthe gene. This step also involves a proofreading mechanism that can replacean incorrectly added RNA molecule.

17.3 TERMINATION:Bacteria use two different strategies for transcription termination: in

Rho-independent transcription termination, RNA transcription stops whenthe newly synthesized RNA molecule forms a hairpin loop, followed by a runof Us, which makes it detach from the DNA template. In the “Rho-dependent”type of termination, a protein factor called “Rho” destabilizes the interactionbetween the template and the mRNA, thus releasing the newly synthesizedmRNA from the elongation complex. Transcription termination in eukaryotesis less well understood. It involves cleavage of the nascent transcript, followedby template-independent addition of As at its new 3' end, in a process calledpolyadenylation.

17.4 PROKARYOTIC VS. EUKARYOTIC TRANSCRIPTION:Prokaryotic transcription occurs in the cytoplasm alongside translation.

Eukaryotic transcription is primarily localized to the nucleus, where it isseparated from the cytoplasm (where translation occurs) by the nuclearmembrane.

17.5 MEASURING AND DETECTING TRANSCRIPTION:-Transcription can be measured and detected in a variety of ways:• Northern blot• RNase protection assay• RT-PCR• In vitro transcription• In situ hybridization• DNA microarrays

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17.6 TRANSCRIPTION FACTORIES:Active transcription units are clustered in the nucleus, in discrete sites

called ‘transcription factories’. Such sites could be visualized after allowingengaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labeling the tagged nascent RNA. Transcriptionfactories can also be localized using fluorescence in situ hybridization, ormarked by antibodies directed against polymerases. There are ~10,000factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymeraseII factories and ~2,000 polymerase III factories. Each polymerase II factorycontains ~8 polymerases. As most active transcription units are associatedwith only one polymerase, each factory will be associated with ~8 differenttranscription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory.

17.7 TRANSCRIPTION INITIATION COMPLEX:Transcription factors mediate the binding of RNA polymerase and the

initiation of transcription. The RNA polymerase only binds to the promoterafter certain transcription factors are assembled. The completed assembly oftranscription factors and RNA polymerase bound to the promoter is called thetranscription initiation complex.

History: A molecule which allows the genetic material to be realized as aprotein was first hypothesized by Jacob and Monod. RNA synthesis by RNApolymerase was established in vitro by several laboratories by 1965; however,the RNA synthesized by these enzymes had properties that suggested theexistence of an additional factor needed to terminate transcription correctly.Recently, Roger D. Kornberg won the 2006 Nobel Prize in Chemistry “for hisstudies of the molecular basis of eukaryotic transcription”.

17.8 TERMINOLOGY:Activator is a DNA-binding protein that regulates one or more genes by

increasing the rate of transcription. Repressor is a DNA-binding protein thatregulates one or more GE-VIL alpha & beta gene by decreasing the rate oftranscription. Upstream, denotes the region to the left of the +1 (or towardsthe 5' end) transcription initiation site. Downstream, denotes the region tothe right (or towards the 3') of the termination site. Thus, transcription of asingle gene follows this pattern: 5'- promoter - +1 transcription initiation site- RNA-coding sequence - terminator - transcription termination site - 3'<—upstream downstream —>

17.9 REVERSE TRANSCRIPTIONSome viruses (such as HIV, the cause of AIDS), have the ability to transcribe

RNA into DNA in order to see a cell’s genome. The main enzyme responsiblefor this type of transcription is called reverse transcriptase. In the case of

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HIV, reverse transcriptase is responsible for synthesising a complementaryDNA strand (cDNA) to the viral RNA genome. An associated enzyme,ribonuclease H, digests the RNA strand and reverse transcriptase synthesisesa complementary strand of DNA to form a double helix DNA structure. ThiscDNA is integrated into the host cell’s genome via another enzyme (integrase)causing the host cell to generate viral proteins which reassemble into newviral particles. Subsequently, the host cell undergoes programmed cell death(apoptosis).

17.10 LET US SUM UPTranscription is the process through which a DNA sequence is

enzymatically copied by an RNA polymerase to produce a complementary RNA.

The stretch of DNA that is transcribed into an RNA molecule is called atranscription unit.

It contains three stages initiation, elongation and termination.

Transcription may be measured and detected by northern blot, RNaseprotection assay, RT-PCR, In situ hybridization, and DNA microarrays.

17.11 POINTS FOR DISCUSSIONElaborate the role and significance of transcription.

17.12 CHECK YOUR PROGRESSDescribe the initiation and elongation of DNA transcriptionNote: a) Please don’t proceed till you attempt the above question.


17.13 LESSON-END ACTIVITIES1) What is DNA Transcription2) Define the terms initiation, elongation and termination.

17.14 REFERENCES



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128

LESSON - 18

TRANSLATION

CONTENTS


18.1 Initiation

18.2 Elongation

18.3 Termination

18.4 Let us Sum up




18.8 References

18.0 AIMS AND OBJECTIVESTo learn and understand the process of translation

Translation occurs in the cytoplasm where the ribosomes are located.Ribosomes are made of a small and large subunits which surround themRNA.Transfer RNA (tRNA) molecules are 75 - 95 nucleotides long and havefour arms and three loops. True to its name, tRNA transfers amino acids tothe site of the growing protein chain (polypeptide). Each tRNA moleculerecognises a specific, three base-pair mRNA code or codon (the DNA form of acodon is called a triplet and the sequence on the tRNA is called an anticodon).Since there are three bases and four possible nucleotides, there can be up to64 (4x4x4) possible tRNA molecules. Three of these tRNA molecules recognise“stop” or termination codons which have been named amber (UAG), opal (UGA)and ochre (UAA).

The codon indicates which amino acid is to be added and the amino acidis attached to the tRNA molecule at the acceptor arm. As we can see from thetable below, most amino acids are represented by more than one codon. Thismeans that the expected protein can still be synthesised, even when a degreeof mutation occurs in the DNA or mRNA. There are 20 essential amino acids;however they can be combined in any order, just like the four nucleotides.This permits the production of the many different proteins which let organismsgrow and function.

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18.1 INITIATION:When the large ribosmal subunit, small ribosomal subunit, mRNA and

the tRNA carrying a methionine come together in the cytoplasm, the ribosomebecomes active and the synthesis of a polypeptide, or “translation”, is initiated.The AUG codon binds at the protein binding site (P) of the ribosome and AUGis always the first codon of an mRNA the next complementary tRNA will bindat the attachment binding site (A) of the ribosome. The adjacent amino acidsare then joined by a peptide bond via a peptidase enzyme. Thus the polypeptidechain begins to grow and as it does, it is passed to the next tRNA currentlyoccupying the A site.

18.2 ELONGATION:The ribosome then moves 1 codon down the mRNA in a 5' to 3' direction.

This is achieved by a translocase enzyme. As the process of ribosometranslocation continues, the “old” tRNA is released to bind another aminoacid and go in search of a new codon. The binding of a new aminoacid ismediated by an enzyme called amino-acyl-tRNA synthase.

Fig. 46 : Translation

TERMINATION:The process continues along the mRNA until a stop codon is reached.

While there is no tRNA for a stop codon, there is an enzyme called releasefactor which cleaves the polypeptide chain resulting in a new protein.

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Finally, the entire complex is disrupted, the ribosome separates and themRNA is released to be used again or degraded. Translation occurs at multiplesites along an mRNA so that many ribosomes can be seen by electron microscopybound to a single mRNA strand with many polypeptide chains forming fromeach. Not only do different sequences make different proteins, but slightsequence changes can radically change the shape of a protein. The shape orstructure of the protein is essential for its correct function e.g. as an enzymeor an ion channel embedded in a cell membrane

18.4 LET US SUM UPTranslation is a process of synthesis a protein.Translation occurs in the cytoplasm where the ribosomes are located.It contains three stages, initiation. elongation and termination.Elongation is achieved by the translocase enzyme.The process of elongation continues along the mRNA until a stop codon is

reached and this is called as termination.Points for Discussion“Translation is a process of synthesis of a protein” – Elaborate and justify.

18.6 CHECK YOUR PROGRESSWhat are the enzymes involved in elongation and termination of DNA

translation?Note: a) Please don’t proceed till you attempt the above question.


18.7 LESSON-END ACTIVITIES1) Describe the mechanism of DNA translation.

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18.8 REFERENCES



3. Shanmughavel, P. 2005, Principles of Bioinformatics, Pointer Publishers,Jaipur, India.


Documents

Fundamentals of Biological System