PURIFICATION AND CHARACTERIZATION OF

ASCARIS SUUM ALDOLASE: AN INITIAL

PHYLOGENETIC STUDY OF ALDOLASES

APPROVED:

Major Professor

/ // uL •IS-Minor Professor

*) xKiDt i. Chairmanofthe Department of Biology

Dean Uf the Graduate School "

i?/ i /

Dedman, John R., Purification and Characterization of

Ascaris suum Aldolase: An Initial Phvlogenetic Study of

Aldolases. Master of Science (Biology), August, 1972, 74

pp., 10 tables, 12 figures, 101 titles.

An efficient purification procedure of Ascaris suum

muscle utilizing ion exchange column chromatography has been

developed. Homogeneity of the enzyme preparation was con-

firmed by rechromatography, ultracentrifugation, and disc

gel electrophoresis. The molecular and catalytic properties

of the enzyme are comparable to other muscle aldolases. The

molecular weight, determined by ultracentrifugation and gel

filtration, is 160,000 composed of four similar subunits.

The sedimentation coefficient, diffusion coefficient, fric-

tional ratio, Stokes' radius, amino acid composition, and

extinction coefficient have been determined. The Michaelis

constants for fructose-1,6-diphosphate (FDP) and fructose-1-

phosphate (F1P) are 6 x 10"" ̂ M and 3 x 10~2 M, respectively.

The FDP/F1P ratio is 42.

Tryptic peptide fingerprints and amino acid analysis

Ascaris and rabbit muscle aldolases also exhibited a high

degree of homology. It appears that muscle aldolases have

been very conservative in maintaining their primary structure.

A proposal of a phylogenetic study of aldolases utilizing a

quantitative method of comparing proteins from their amino

acid compositions is discussed.

PURIFICATION AND CHARACTERIZATION OF

ASCARIS SUUM ALDOLASE: AN INITIAL

PHYLOGENETIC STUDY OF ALDOLASES

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

John R. Dedman, B. S

Denton, Texas

August, 1972

"It is quite conceivable that every species tends to produce varieties of a limited number and kind, and that the effect of natural selection is to favour the development of some of these, while it opposes the development of others along their predetermined lines of modification."

Thomas H. Huxley Evolution in Biology. 1878

INTRODUCTION

Aldolase (E. C. 4.1.2.13 f ructose-1,6-diphosphate-

D-glyceraldehyde-3-phosphate lyase) provides an essential

role in the glycolytic pathway of all organi&ms, with the

exception of some heterofermative bacteria (Gunsalus, 1952).

It catalyzes the cleavage of fructose-lj6-diphosphate (FDP)

to the trioses, dihydroxyacetone phosphate (DHAP) and glycer-

aldehyde-3-phosphate (G-3-P) (reaction l).

HS-0-P03

c=o

H HOCH HC-O-PO, HC=0

I I , I H C 0 H - Aldolase . C=0 + HCOH i I i

HCOH HCOH HC-0-P0o I H H 3

HC-O-PO-H 3

FDP DHAP G3P

Although the equilibrium constant is in favor of FDP

formation, the reaction is readily reversible; when there is

sufficient removal of the triose products during glycolysis.

Aldolase has an absolute specifity for DHAP (Morse and Horecker,

1968), although a wide variety of aldehydes and their ketoses

(phosphorylated or non-phosphorylated) including L-glycer-

aldehyde, D and L-erythrose, D and L-threose, formaldehyde

acetaldehyde, glycolaldehyde, and propionaldehyde may constitute

the remaining carbon requirement (Morse and Horecker, 1968}

Rutter, I960: Horecker, 1959)•

Aldolase activity was first demonstrated by Meyerhof and

Lohmann (1934)> who believed the enzyme to also contain the

catalytic function of triose phosphate isomerase. This

activity was found to be a contaminant when extensively puri-

fied by Herbert (1940). Warburg and Christian (1943) recorded

the first crystallization of the enzyme from rat muscle and

yeast.

Two distinct forms of aldolase have been reported in

biological systems. Rutter (1964) classified these unique

forms as Class I and Class II, each possessing strikingly

diverse physical and catalytic properties. Class II aldolase

is restricted to bacteria, fungi, yeast, and blue-green algae.

Its most distinguishing characteristics are a requirement

of a divalent metal ion and a molecular weight of 80,000

(Rutter, 1964j Harris et al., 1969; Kobes et al., 1969;

Mildvan et al., 1971).

Class I aldolases are present in animal and higher plant

tissues. They exist as tetramers of molecular weight 160,000. I

The exception is spinach leaf aldolase, which has a molecular

weight of 120,000 (Fluri et al., 1967). Class I aldolases

are not affected by metal chelators. They form a Schiff base

intermediate between DHAP and the £-amino group of a lysine

residue in the active site (Grazi et al., 1962; Morse and

Horecker, 1968: Rutter, 1964; Horecker, 1970). The comparative

properties of Class I and Class II aldolases are summarized

in Table I. Euglena and Chlamydomonas (Rutter, 1964) possess

both forms of aldolase, Class II during heterotrophic growth,

and Class I during autotrophic phases.

The animal aldolases have shown variformity in tissue

localization (Penhoet et al., 1966; Masters, 1967; Rutter

et al., 1968; Masters, 1967} 1968; Lebherz and Rutter, 1969J

Kochman et al., 1971? Penhoet et al., 1 9 6 9 a ) . There are three

types, A, B, arid C, characteristic of muscle, liver, and brain

aldolase, respectively (see Table II). The three types are

similar in physical properties, including molecular weight,

subunit structure, and catalytic mechanism forming a Schiff-

base intermediate (Morse and Horecker, 1968a; Penhoet et al..

1969b: Schachman et aj.., 1966; Penhoet et al., 1967; Sia et

al.. 1968; Kawahara et al., 1966; Gracy et al., 1 9 6 9 ) .

However other parameters exhibit significant differences.

Distinctions may be seen in immunological cross-reactions

(Penhoet et al., 1966; Penhoet et al., 1969; Penhoet and

Rutter, 1971; Marquart, 1971; Baron et al., 1969), amino-acid

compositions (Gracy et al., 1970), tryptic fingerprints (Gracy I

St J 1970; Penhoet et al., 1969b; Rutter, 1 9 6 4 ) ,

sequences of amino-acids in the active sites (Morse and

Horecker, 1968b), and end-group analysis (Ting et al., 1971;

Lai and Oshima, 1969). These properties are summarized in

Table III.

The major catalytic distinctions between the Class I

TABLE I

PROPERTIES OF ALDOLASE CLASSES

Class I Class II

Animals, plants, green algae, euglena and chlamydomonas (Autotrophic)

Schiff-base intermediate ((-amino lysine-DHAP)

Functional carboxyterminal tyrosine

Broad pH profile

MW 120,000-160,000

s 20 ,» 6 .5 -8 .0

4 subunits

FDP/FIP ratio 50,1,10

Specific Activity 8r25

Bacteria, yeast, fungi, blue-green algae, euglena &1 chlamy-domonas (Heterotrophic)

Divalent metal ion requirement K + activation

Functional SH groups

Sharp pH profile (7»0-7.5)

MW 70-80,000

S20,w 5-5-6'.0 S

2 subunits

FDP/F1P ratio 2000-2500

Specific Activity 80-100

From: Rutter, 1964* Rutter et al., 1968.

TABLE II

TISSUE DISTRIBUTION OF ALDOLASE ISOZYMES

Tissue Isozyme

Muscle

Heart

Spleen

Kidney cortex medulla

Brain

Liver

^-Intestine

*Lens

A

A

A

A-B A

A-C

A-B

A, B

C

From: Lebherz and Rutter, 1969*

•frKochman et al., 1971.

TABLE III

PROPERTIES OF CLASS I ALDOLASE VARIANTS (RABBIT)

Properties A B C

^Molecular Weight 160,000 160,000 160,000

#Schiff-base Intermediate + + +

°Immunological cross-reactivity

Anti-A Anti-B Anti-C

+ +

+

^Residual act-ivity after carboxypep-tidase A 5% 55% 1056

w^max (FDP Cleavage) (FDP Synthesis)

2,900 10,000

250 2600

1,000 4,900

*Km (FDP) 3 X 10~6

1 x 10~9 3 x 10-6

( F IP ) 1 x 10""2 3 x 10-4 4 x 10"3

(DHAP) 2 x 10~3 4 x 10-4 3 x 10-4

(G3P) 1 x 10-3 3 x 10-4 8 x 10"4

*FDP/F1P Ratio 50 1 10

#Rutter et al., 1968. " " °Penhoet, Kochman, and Rutter, I969, *Penhoet and Rutter, 1971.

aldolases are the maximum velocities of cleavage of the two

substrates (ie, FDP/F1P ratio). Mammalian muscle (type A),

liver (type B), and brain (type C) possess FDP/F1P ratios of

50, 1, 10, respectively (Penhoet et al., 1966; Penhoet et al..

1 9 6 9 b ) . The physiological function of type A and B aldolases

have been correlated with these catalytic properties. The

muscle form functions primarily in glycolysis, and the muscle

does not utilize F1P as a substrate. On the other hand, the

liver can convert fructose into F1P by a specific fructokinase

which in turn is cleaved by liver aldolase. The glyceraldehyde

formed from this reaction can be phosphorylated by a specific

triose-kinase. Likewise, ATP is a competitive inhibitor of

the muscle type and the liver enzyme is strongly inhibited

by AMP (Spolter et al., 1965; Gracy et al., 1970). Both types,

A and B, fulfill physiological function in the specific tissues

(Rutter et al., 1963; Horecker, 1967) • The specialized func-

tion of brain aldolase has yet to be elucidated (Herskovits

et al., 1 9 6 7 ) .

The ontogeny of aldolase has been studied by examining

the isozyme patterns in specific tissues (Rutter et al., 1963;

Weber and Rutter, 1964; Sheedy and Masters, 1971; Masters,

1 9 6 8 ) . In most tissues, mixed hybrid forms are originally

present, and not until the tissue develops and differentiates

do the parental forms become predominant (Masters, I 9 6 8 )

(Table II). Adult muscle maintains type A and liver is pre-

dominately type B, while kidney and brain possess the five

8

respective A-B and A-C hybrids (Penhoet et al., 1966; Rutter

et al., 1968; Kochman et al., 1971; Lebherz and Rutter, 1969).

Although ABC and BC hybridization has been produced in vitro,

such combinations have yet to be found in biological systems

(Penhoet et al,, 1966; Penhoet et ajL., 19<?7). Recently,

Penhoet and Rutter ( 1971) examined the catalytic and immu-

nological properties of aldolase variants. An-Cn and

Bn-Cn mixtures possessed proportional FDP/F1P ratios indi-

cating that, catalytically, the subunits act independently.

This has also been verified by the hybridization experiments

using succinic anhydride inactivated subunits (Meighen and

Schachman, 1 9 7 0 ) . Anti-sera against A does not react with

B or C and vice versa. But anti-sera from A, B, or C inhibit

activity of any combination of its hybrids.

Because of its omnipresence in biological systems and

relative ease of purification, aldolase has been extensively

studied and amply characterized. This has created an area

of extensive information and interest in comparative bio-

chemistry of living organisms. Until now, aldolase has been

purified and studied in a large variety of vertebrates

(Anderson et al., 1969; Caban and Hass, 1971; Ikehara et al..

1 9 6 9 ) , spinach (Fluri et al.., 1967; Rapaport et al., I969),

and microorganisms (Kobes et al., 1969; Mildran et al., 1 9 7 1 ) .

Although this study encompasses a wide variety of organisms,

there is a large void of information in the invertebrate

classes, the lobster (Buha et al., 1971) being the lone

species studied. This has resulted in a strong need for a

thorough investigation of the invertebrates.

The present study of Ascaris aldolase is twofold; first,

from a comparative biochemical standpoint, and secondly from

a clinical aspect. There is a need for more efficient anthel-

mintic drugs. From recent studies, many drugs have been found

to inhibit specific Ascaris enzymes (Saz and Bueding, 1966;

Bueding, 1969; Saz and Lescure, 1968; Van den Bossche and

Jansen, 1969). The greater our knowledge of the differences

between the parasite's and host's metabolic pathway^ the more

confident we can be of a vermifuge's effectiveness. There

are marked differences in the metabolic pathways of the worm

and host (Saz and Lescure, 1969; Saz, 1971; 1971b). Although

helminth enzymology is in its infancy, a few reports of the.

properties of purified helminth enzymes have recently appeared

(Burke et al., 1972; Li et al., 1972; Fodge et al., 1972; Hill

et al., 1971). In addition, partial purification of nematode

aldolases has been reported (Mishra et al., 1970; Srivastava

et al., 1971).

The present pcper describes the purification and properties

of the enzyme aldolase from the muscle tissue of the pig intes-

tinal roundworm, Ascaris suum. In addition, the properties of

the ascarid enzyme have been correlated with muscle aldolases

of other organisms in an attempt to evaluate the evolutionary

development of the enzyme.

MATERIALS AND METHODS

Materials

— 1

Ion exchanger, cellulose phosphate (0.91 meq g ), the

sodium salts of NAD, NADH, fructose-1,6-diphosphate (FDP);

2-mercaptoethanol, ammonium sulfate (enzyme grade), crystalline

bovine serum albumin, ̂ -glycerol phosphate dehydrogenase,

triose phosphate isomerase, ethylenediaminetetracetic acid

(EDTA), phenazine methosulfate, MTT tetrazolium 3(4,5-

dimethylthiazolyl-2-)-2-5-diphenyl tetrazolium bromide,

triethanolamine, and the barium salt of fructose-l-phosphate

(F1P) were obtained from Sigma Chemical Company. Guanidinium

chloride, sodium dodecyl sulfate (SDS), and ninhydrin were

1Sequanal grade' and purchased from Pierce Chemical Company.

Sephadex G-200, aldolase, chymotrypsin, albumin, and ovalbumin

were acquired from Pharmacia Fine Chemicals. Aldolase and

glyceraldehyde-3-phosphate dehydrogenase, crystallized from

rabbit muscle, and TPCK-treated trypsin were prepared by

Worthington Chemicals. Iodoacetic acid (Eastman Organic

Chemicals) and urea (Mallinckrodt) were recrystallized prior

to use. The barium salt of F1P was converted to the sodium

salt with Dowex50 (Mesh 200-400). All other chemicals were

of reagent grade and all solutions were prepared in distilled

water.

10

11

Enzyme Assay

Aldolase was assayed according to the coupled enzyme

procedure of Racker (1947). Assays were performed in a

temperature—rfegulated Beckman Model DB—GT Spectrophotometer

attached to a Beckman Ten-inch Laboratory Potentiometric

Recorder. Initial velocities were determined by monitoring

the linear decrease in the absorbance of oxidized NADH at

340 nm at 25°C. The routine reaction solution consisted of

130 yumoles triethanolamine buffer (pH 7»5)> 0.75 xunoles

NADH, 3.0 jumoles FDP, and 10 xig each of triose phosphate

isomerase and ©^-glycerophosphate dehydrogenase in a 3 ml

final volume. One unit of activity is defined as the amount

of enzyme catalyzing the conversion of 1>umole of FDP per

minute at 25°C, Specific activities are expressed as units

per mg protein. During kinetic studies, precise measurements

were made by observing the optical change from 0.0-0.1 0D

with a chart speed of 10 inches per minute. Substrate con-

centrations were determined spectrophotometrically.

Protein Determination

The protein concentration of crude homogenate was deter-

mined by the Biuret method (Bailey, 1967) using a standard

curve of 1-5 mg ml""*- crystalline bovine serum albumin. All

other protein estimations were determined from absorption at

280 nm using the molar absorbancy index of 1.06.

12

Ion-Exchange Chromatography

Cellulose phosphate was washed repeatedly with 0.5 N

HC1 and 0.5 N NaOH until the filtrate remained colorless

(Peterson and Sober, 1962). The exchanger was then equil-

ibrated in 10 inM triethanolamine, 1 mM EDTA, and 10 mM

2-mercaptoethanol at the desired pH and stored at 0-5°C.

A suspension of approximately 1 gm per 15 ml buffer was allowed

to de-aerate under vacuum with vigorous stirring. Columns were

packed under hydrostatic pressure and washed with buffer (50

ml hr-l) at 5°C for 24 hours. The eluant was tested for the

desired pH before the sample was applied. Constant flow rates

(50 ml hr ) were maintained with a piston minipump. Fractions

of 10 ml were collected. Protein was monitored by the absor-

bance at 280 nm.

Gel Filtration

Sephadex G-200 (particle size 40-120 AJL) was equilibrated

in 5 mM phosphate buffer suggested by Pharmacia (1970) and

packed under 10 cm hydrostatic pressure. The 1.6 x 120 cm

column was washed with the buffer at 5°C until stabilization

of bed height and a constant flow rate of 6 ml hr""l were

achieved. Samples in 20% sucrose (w/v) were layered onto the

top of the column bed. Dimensions of the column, void volume

(VQ), internal volume (V^), and elution volumes (Ve) were

determined with blue dextran, tryptophan, and standard proteins

(Pharmacia). The elution volumes were correlated with molec-

ular weights (Andrews, 1964j Determann and Michel, 1966) or

13

Stokes' radii (Andrews, 1970).

Polyacryamide Gel Electrophoresis

Disc gel electrophoresis was conducted according to the

procedure of Davis (1964)- Gels were prepared with a 7*5%

monomer concentration in Tris-glycine buffer (pH 8.9) and

run on a Canalco Model 200 apparatus. Sodium dddecyl sulfate

gel electrophoresis was performed following the procedure of

Weber and Osbox-n (1969), using 10$ gel in 0.1% SDS and phosphate

buffer (pH 7.0). The mobilities of the protein subunits

were plotted against their molecular weights. Protein was

stained with 0.25$ (w/v) Coomassie Brilliant Blue in methanol:

acetic acidsI^O (5:5:90). Activity bands were developed via

the method of Lebherz and Rutter (1969), coupling glyceralde—

hyde-3-phosphate dehydrogenase reduction of NAD with phenozine

methosulfate and MTT tetrazolium.

Ultracentrifugation

Sedimentation velocity and sedimentation equilibrium

experiments were conducted in a Beckman-Spinco model E ana-

lytical ultracentrifuge equipped with RTIC temperature and

electronic speed control. Schlieren patterns and Rayleigh

interference fringes were measured with a Nikon model 60

microcomparator with digital encoders. High-speed sedimen-

tation equilibrium experiments were carried out as described

by Yphantis (1964) and Van Holde (1967). Densities and vis-

cosities of all buffers were calculated as described by Rozacky

14

et al. (1971) and Kawahara and Tanford (1966). The partial

specific volume was estimated from the amino-acid composition.

Tryptic Fingerprints

Samples of purified aldolase (10-20 mg) were S-carbox-

ymethyiated in 10 ml of 8M urea, 0.2% EDTA, 0.1 ml 2-mercap-

toethanol, and 0.268 g iodoacetic acid (Scoffone and Fontana,

1970). A 1/50 weight ratio of TPCK treated trypsin was used

to digest the aldolases at pH 8.0 in 2.5% trimethanolamine.

After dialysis in y% formic acid and lyophilization, the

samples were diluted to a concentration of 10 mg ml-"'". Approx-

imately 200 jag of protein was carefully spotted near the

corner of a 20 x 20 cm celluose-coated (160 microns) thin-

layer plate (Eastman Organic Chemicals). Best results were

obtained when electrophoresis was carried out first at 300

V for 2 hrs under varsol in pyridine:acetic acid:H20 (100:

30:3000) pH 5.5 at 5°C. The plate was then chromatographed

in the second dimension in n-butanol:pyridine : acetic acid:

^2® (150:100:30:120) to 1 cm of the upper edge. After the

dried chromatogram was sprayed with 2% ethanolic ninhydrin

with 10$ collidine, it was allowed to develop for 30 min in

a 75°C oven (Tarui et al., 1972). The colored spots were

outlined and the plates stored at 4°C with little loss in

intensity for several weeks.

Amino Acid Analysis

One milligram samples were hydrolyzed for 24 and 48 hrs

15

in sealed evacuated tubes containing 2.0 ml of 6N HC1 at

110°C (Moore and Stein, 1963). Norleucine was used as an

internal standard. Analyses were performed on a Beckman

model 120C automatic amino acid analyzer (Spackman et al.,

1958). Cysteine was determined from the S-carboxymethyl

derivative (Scoffone and Fontana, 1970) and tryptophan was

estimated spectrophotometrically (Edelhoch, I967).

RESULTS

Purification

Female Ascaris were obtained from a local abattoir.

Muscle tissue was dissected free from reproductive and di-

gestive organs then, washed in cold buffer. The muscle was

then homogenized in 3 volumes of 20 mM triethanolamine, 0.1

M NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol (pH 7.5) for three

1-min intervals with 5—min intermittent cooling periods. The

mixture was centrifuged for 1 hr at 12,000 x g in a refrigerated

centrifuge at 0-5°C. The resulting pellet was rehomogenized

for 1 min in 1 volume of buffer and recentrifuged as above.

The supernatant solutions were pooled and again centrifuged

at 90,000 x g for 1 hr (Beckman model L 3-40) and strained

through glass wool to remove any residual lipid. The resul-

tant pale-pink solution ('Extract') was then allowed to dialyze

against 4 liters 10 mM triethanolamine, 1 mM EDTA, 10 mM 2-

mercaptoethanol buffer (pH 7.1) for 24 hrs with three changes

of buffer.

The dialized fraction was applied to a column (2.5 x 45

cm) of phosphocellulose and washed with the respective buffer

until the eluant was protein-free. Subsequently, a linear salt

gradient (0.0-0.5M NaCl; 500 ml to 500 ml) was begun (Fig. l),

Fractions containing aldolase activity (Fig. 1) were combined

('Salt Elutant') and dialyzed 20 hrs against the same buffer

16

17

Fig. 1. Column chromatography of Ascaris aldolase on phosphocellulose. 'EXTRACT' (480 ml) was applied to a phos-phocellulose column (2.5 x 60 cm). Buffer (10 mM trieth-anolamine 10 mM 2-mercaptoethanol, ImM EDTA, pH 7*1) was pumped through until the eluate was free of protein. A linear salt-gradient was employed starting with buffer (500 ml) in the mixing chamber and 500 ml of 0.5M NaCl buffer solution in the reservoir. Arrow designates the initiation of the gradient. The major peak of activity was pooled ('SALT ELUTANT') and dialyzed in the same buffer at pH 7»4«

( t _ | i u 6 u i ) N i 3 x o a d

lp

? O s o o n o i o ^ P ) N » -

CO CN i- j o o d o

•—• ( . [ U I ^ U I U i p O A D a Q d a d S ® | O W 9 - 0 t

J UJ % 3 —J

o >

19

at pH 7.4. The sample was applied to a second phospho-

cellulose column (1.5 x 90 cm) equilibrated in the appropriate

buffer. Again the non-binding protein was washed free from

the column. A 2.5 mM fructose-1,6-diphosphate solution (in

the same buffer) was applied eluting all of aldolase activ-

ity in a single, sharp peak in which the activity and protein

fractions coincided ('FDP Elutant', Fig. 2). The purified

enzyme solution was immediately dialyzed for 15 hrs in satur-

ated ammonium sulfate to avoid loss of activity (Woodfin and

Temer, 1967)• The precipitated protein was collected by cen-

trifugation at 25,000 x g.

The total purification of Ascaris muscle aldolase (Table

IV) is 157-fold, a value consistent to that reported for other

muscle aldolases. A final yield of 11.2 mg enzyme represented #

67% recovery of activity from an original 130 gm of Ascaris

muscle. A specific activity of 10.2 can be compared with other

type A aldolases, frog, 11.5 (Ting et al., 1971), shark, 25.5

(Caban and Hass, 1971), tuna, 7.5 (Kwon and Olcott, 1968),

lobster, 14.8 (Guha et al., 1971), chicken, 24 (Marquart, I969),

codfish, 8 (Lai and Chen, 1971), and rabbit, 12 (Schwartz and

Horecker, 1966). Comparable results were obtained with several

other preparations, and the precipitated enzyme was pooled and

stored at 4°C in 70% ammonium sulfate.

The absorbance of a 0.1% solution of Ascaris aldolase at

280 nm is 1.06, This is consistent with the greater number of

tryptophan residues in the ascarid enzyme (vide infra).

20

Fig. 2, Substrate elution of Ascaris aldolase from phosphocellulose. Partially purified enzyme ('SALT ELUTANT'j 300 ml, 310 mg, specific activity = 0.4) was applied to a phosphocellulose column (1.5 x 90 cm) and washed with buffer until fractions were protein free. A 2.5 mM FDP solution (in same buffer) was then applied (arrow A), specifically eluting aldolase. The purified enzyme solution was immediately dia-lyzed against saturated ammonium suffate. Salt solution (1.0M: B) was applied to elute the remaining protein which was devoid of aldolase activity.

2,1

10~6 Moles FDP Cleaved

min"1 ml"1 • — • __i ro w • • • o o

< O r— c £ m

o o o

o% o o

i t

O Ui

PROTEIN (mg ml""1)

22

TABLE IV

PURIFICATION OF ASCARIS MUSCLE ALDOLASE

Procedure Vol. Protein Units Spec- Yield Fold (ml) (mg) ific % Purif.

Act.

'Extract' 480 2620 170 0 . 0 6 5

'Salt Elutant' 300 310 123 0 . 4 72 6 . 2

'FDP Elutant' 45 H . 2 114 1 0 . 2 67 1 5 7 . 0

23

Protein concentration of the solution was determined by the

method of Lowry et al. (1951)•

Gel Filtration

The molecular size of Ascaris muscle aldolase was also

determined from quantitative gel filtration of a calibrated

Sephadex G-200 column (1.5 x 120 cm; see Figure 3). Chro-

matography of Ascaris and rabbit muscle aldolases on Sephadex

(Fig. 3 and 4) results in coinciding elution peaks, demon-

strating the striking similarity of molecular size of the two

muscle aldolases. The apparent molecular weight of Ascaris

muscle aldolase from gel filtration is 158,000 + 3000.

The Stokes' radius was determined from the gel filtration

data according to the procedure of Ackers (1968). The Stokes'

radius of Ascaris aldolase is 46.4 ^ (Fig. 5). The diffusion

constant was estimated from the relationship D^Q w — KT/6Trtytt, ,

(Siegel and Monty, 1965), where 1 is the Stokes' (46.4 A), K

is the Boltzman constant, and is the viscosity of water at

20°C (0.01005 P). Ascaris aldolase has a diffusion constant n O

of 4.43 x 10 cm /sec. Further, a frictional ratio (f/fQ)

of 1.29 for Ascaris aldolase is indicative of an essentially

spherical molecule. These are essentially the same values as

rabbit muscle, since it was used as a standard.

Folyacrylamide Gel Electrophoresis

Disc gel electrophoresis of Ascaris muscle aldolase re-

sulted in a single band of protein which corresponded to the

24

Fig. 3. Determination of molecular weight of Ascaris aldolase by gel filtration. The logarithm of the molecular weight is plotted against the ratio of protein elution volume (Ve) to column void volume (VQ) . A Sephadex G-200 column (1.5 x 120 cm) was calibrated with (A) chymotrypsin A, MW 25,000, (B) ovalbumin, MW 45>000, and (C) rabbit muscle aldolase, MW 158,000. The open circle (0) represents Ascaris aldolase. Fractions of 40 drops each were collected (flow rate of 6.0 ml per hour).

n

2.75

>° 2.25 >"

1.75

—

\ a

-

S > S « B

mm \ | C

1 1 1 1 30 50 100 150

MOLECULAR WEIGHT

(x lO 3 )

26

Fig. 4. Comparative elutions of Ascaris and rabbit muscle aldolase on Sephadex G-200 column(1.5 x 120 cm). Samples were chromatographed on separate runs.

27

RABBIT MUSCLE ALDOLASE

O D280

io o

73 > n -H o z z c £ CO rn 79

O

a O

CO o

o o

ASCARIS ALDOLASE ACTIVITY

(units ml"1) • — •

28

Fig. 5. Determination of molecular radius of Ascaris aldolase from gel filtration data. values (Ve-V0/V.)are plotted against the Stokes' gadii of CA) chymotrypsin A, 20.9 A, (B) ovalbumin, 27.3 A, and rabbit muscle aldolase, 45 a. The open circle (0) designates Ascaris aldolase.

2$

K.

0 . 4 -

20 SO 4 0 MOLECULAR RADIUS ( A )

30

single band when stained for aldolase activity {Fig, 6), thus

providing another criterion of homogeneity. Electrophoresis

in the presense of sodium dodecyl sulfate (SDS) yielded a

single band. Ascaris aldolase, disassociated in SDS, exhibits

a subunit molecular weight of 42,000 + 3000(Fig, 7).

Ultracentrifugation Studies

As shown in Figure 8,the sedimenting enzyme formed a

single, symmetrical boundary, indicating a homogeneous pre-

paration of the enzyme. Values of the sedimentation coeffi-

cient were calculated from three protein concentrations (2.87,

1.49. 0.74 mg ml~^) and the s2o w w a s °btained by extrapolation

to zero protein concentration. The value of 7.98 S (Fig. 9)

is in the same order as reported for Class I aldolases (Penhoet

al., 1969b; Gracy et al. 1969; Ting et al, 1971; Lai and

Chen, 1971; Guha et al., 1971). The S2o,w m ay be calculated

from the relationship S 2Q j W =® 7.98 (l~0.008c), where c is

the protein concentration in milligrams ml""**'.

Sedimentation equilibrium studies yielded a molecular

weight of 156,000 + 3000. When disassociated in 6M guanidine

hydrochloride the molecular weight is 40,800 + 1000, indicating

a tetrameric structure of similar subunits with virtially

equal mass. Plots of (In y vs x 2) resulted in straight lines,

suggesting a monodisperse system (Fig. 10).

Catalytic Properties

The FDP/F1P ratio for Ascaris muscle aldolase is 42 from

31

Fig. 6. Polyacrylamide gel electrophoresis of Ascaris aldolase. Purified enzyme (50 micrograms with specific activity = 10.2) was applied to a 7.5% standard 0.6 x 7.0 cm polyacrylamide gel. Electrophoresis was carried out at 5° in Tris-glycine buffer (pH 8.9). The gel shown at left was stained for protein,while the gel on the right was devel-oped in a stain specific for aldolase activity ('Methods').

32

33

Fig. 7. Determination of subunit molecular weight on sodium dodecyl sulfate gel electrophoresis. Ascaris aldolase (0.5 mg) and standard proteins were prepared in 1% SDS solution and subjected to electrophoresis on separate gels (10% monomer concentration, 8 ma, 5 hours). The mobility of each protein was plotted against the logarithm of its molecular weight. Letters designate standard proteins, (A) albumin, MW 68,000, (B) ovalbumin, MW 43,000, (C) pepsin, MW 35,000, and (D) trypsin, MW 23,3000.

34

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Fig. 8. Sedimentation-velocity centrifugation of Ascaris aldolase. Purified enzyme (2.87 mg ml"1) was centrifuged in a 12 mm double sector cell at 60,000 rpm at 20°C. The solute sector and reference sector contained 0.40 ml of 10 mM Tris-Cl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 M NaCl, pH 7.5.

36

37

Fig. 9. Determination of sedimentation coefficient of Ascaris aldolase. Purified Ascaris aldolase in concentrations of 2.87* 1«43* and 0.72 mg ml" were subjected to sedimentation velocity experiments at 60,000 rpm. The §20 w

v a l u e (7*985) was obtained by extrapolating to zero protein concentration.

38

* O CN CO

1.0 2.0 3.0

PROTEIN (mg ml4 )

39

Fig. 10. Fringe displacement obtained from sedimentation equilibrium of native Ascaris aldolase. The protein (0.5 mg ml ) was dialyzed against 50 mM Tris-Cl, 0.1 M NaCl, 10 mM 2-mercaptoethanol, pH 8.0. Sedimentation was at 16,000 rpm in the An-D rotor in a 12-mm double sector cell with sapphire windows for 24 hours at 20°C. The protein concentration was calculated from the fringe displacement (y"i-y0) • The abscissa represents the square of the distance (cm) from the center of rotation.

40

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comparisons of the V m a x values. The Michaelis constant for

FDP is 6 x 10~^M and for F1P is 3 x 10~^M at 25°C. The con-

centration range for FDP was 5.18 x 10~^M to 3.11 x 10~^M and

that for FIP was 5.75 x 10~2M to 1.01 x 10-1M. Values were

calculated using a computer program with weighted regression

analysis (Cleland, I963). When dialyzed against 0.1 M EDTA,

there was no loss in activity, and divalent or potassium ions

displayed no augmentation in activity. These results are in

accordance with those reported for all other muscles aldolases

except lobster (Guha et al., 1971), which has a catalytic

ratio of 200. Properties of Ascaris muscle aldolase are

summarized on Table V*.

Peptide Maps

Composite maps were drawn from at least five chromatograms

each for rabbit and Ascaris muscle aldolase (Fig. 11). Locali-

zation of the peptides and comparisons were made by overlaying

a 2-cm grid on the maps. The total number of peptides (35)

from both enzymes was consistent with the predicted number of

arginines and lysines present in the amino-acid composition

and the presence of four identical subunits. Surprising sim-

ilarities were discovered from the fingerprints. Most of the

peptides from each muscle type had nearly identical migrations.

Small differences are present in region 2-3, F-G (Fig. 11),

in which Ascaris aldolase has a few additional peptides. Other

relatively small differences in location can be accounted for

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43

Fig. 11. Tryptic peptide fingerprints of rabbit (i) an<* Ascaris (II) muscle aldolases. S-carboxymethylation, tryptic digestion, electrophoresis, and chromatography were performed as described in 'Methods'. The origin is desig-nated by (*). Each drawing is a composite from five chromatograms (cellulose thin-layer plate).

44

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32

1

(2) CHROMATOGRAPHY

45

by minor intra-peptide changes in composition. This would

result in slight alterations in homologous peptides.

Amino-Acid Analysis

The amino acid composition of Ascaris muscle aldolase

was calculated on a basis of a molecular weight of 160,000.

In comparing Ascaris aldolase to the amino acid compositions

of rabbit, the mammalian muscle prototype (Ting et al(, 1971a),

and the pig (Anderson et al^, 1969), significant differences

were found (Table VX ). Table VII lists the major differences

of 10% or greater.

Also as shown in Table VI , the number of hydrophilic

amino acids (lys, arg, his, asp, glu) varies by just 6%. Further,

the hydrophobic amino acids (val, ile, leu, phe) differ by a

mere . The greatest change seems to be a very conservative

substitution of glycine. Ascaris has 29 and 21 more residues

than pig and rabbit, respectively. It is evident that Ascaris

muscle aldolase differs only slightly overall from both rabbit

and pig muscle aldolase.

TABLE VI

AMINO-ACID ANALYSIS RESIDUES PER MOLE

46

Ascaris Muscle

Piga

Muscle Rabbit'3

Muscle

Lysine 112 110 104

Histidine 46 39 44

Arginine 52 56 56

Aspartic Acid 112 116 116

Threonine 82 81 88

Serine 94 79 84

Glutamic Acid 180 162 164

Proline 85 74 80

Glycine 145 116 124

Alanine 162 156 . . 172

HaIf-cystine 30 28 32

Valine 75 84 80

Methionine 18 15 12

Isoleucine 73 77 76

Leucine 118 138 140

Tyrosine 47 45 48

Phenyalanine 39 30 28

Tryptophan 20 12 12

Total 1490 1418 1460

Corrected to 160 ,000 MW.

aValues from Anderson et al,(1969). b Values from Ting et al. ( 1971a) ,

#

47

TABLE VII

COMPARISON OF AMINO-ACID COMPOSITIONS

Amino Acid Pig Rabbit

Amino Acid Residues Different

% Difference

Residues Different

% Difference

Histidine +7 15 +2 4

Serine +15 16 + 10 11

Glutamic acid +18 10 +16 9

Glycine +29 20 +21 .14

Proline +11 13 +5 6

Valine -11 11 -5 1

Methionine +3 17 +6 33

Leucine -20 14 -22 16

Phenylalanine +9 23 +11 28

-^Tryptophan +8 40 +8 40

*Not included in deviation function calculation.

DISCUSSION

Fructose diphosphate aldolase was purified 157-fold

from the muscle of the roundworm, Ascaris suum. The pur-

ified enzyme exhibited a specific activity of 10.2 units

per mg of protein. The preparation was shown to be homoge-

neous by the criteria of analytical ultracentrifugation,

zone electrophoresis, and rechromatography. Ascaris aldolase

is composed of 4 identical subunits of 40,000 daltons each.

Aldolase was partially purified from the nematodes

Ascaris suum (Mishra, 1970) and Ascaridia galli (Strivastava

et al.. 1971). Mishra (1970) reported that Ascaris aldolase

exhibited a FDP/F1P ratio of 82. In addition, ATP, ADP,

and AMP exhibited positive allosteric effects of the enzyme,

which is in contrast with other studies of muscle aldolase

(Spolter et al., 1965; Lacko et al., 1970; Kawabe et al..

I969). The allosteric effects reported by Mishra may be due

to contamination of his preparation with other enzymes such

as phosphofructokinase. Srivastava et al. (1971) purified

Ascaridia galli aldolase 44-fold and recorded a K m value for

FDP of 4»5 x 10~3 M,100-fold higher than any other muscle

aldolase. Due to the inconsistency of these workers' results,

their heterogeneous preparations, and their methods of assaying

the enzyme, their resulting data must be considered tenuous

at this time.

48

49

The present study involved a comparison between the primi-

tive invertebrate, Ascaris suum. and mammalian (rabbit) muscle

aldolase. The molecular weights, sedimentation coefficients,

Stokes' radii, tryptic fingerprints, and amino-acid compositions

are surprisingly similar. Catalytic studies also supported

these similarities. Further, other studies from this labora-

tory (Dedman et al., 1972) suggested that Ascaris aldolase,

like rabbit muscle aldolase, is inhibited by glyceraldehyde-

3-phosphate and also responds to reversible, dark inactivation

by pyridoxal phosphate. The enzyme is also desensitized by

pyridoxal phosphate in the presence of light. Likewise,

Ascaris aldolase is inactivated in the presence of sodium

borohydride and FDP. Finally, Dedman et al. (1972) demon-

strated identical migration of Ascaris and rabbit muscle al-

dolases during cellulose-acetate eletrophoresis.

There are several obvious functional components that are

maintained through the phylogeny of muscle aldolase. The act-

ive site peptide retains essentially all of its primary struc-

ture (vida infra). Every muscle aldolase has an amino-terminal

proline and alanyltyrosine-terminating carboxyl end (Caban

and Hass, 1971)^ and the tyrosine is essential in the cleavage

of FDP (surprisingly, the removal of a single C-terminal

tyrosine decreases the Kffl by 100-fold (Gracy et al., 1970).

Furthermore, the amazing similarity of the tryptic fingerprints

Ascaris and rabbit muscle aldolase stresses the conservatism

in the structure of the enzyme. Many of the alterations in

50

proteins (especially enzymes) do not occur randomly. Folding,

interaction between the subunits and most important, pre-

servation of the active center must be maintained. Radical

changes (hydrophobic for hydrophilic) or even conservative

replacements might alter the catalytic properties of the enzyme.

In observing the amino-acid compositions of animal al-

dolases, there is a high degree of homology. Major variations

from average y^lues exist in lobster, having 136 • lysines (25

higher), 132 alanines (25 fewer), and in Ascaris. with 180 glu-

tamic acids (20 higher) and glycines (25 higher). The half-

cystine residues have been maintained at approximately 30

residues per mole, with the exception of codfish (16) and lobster

(12). The most variant amino-acid is methionine, ranging from

4 (frog) through 32 (codfish) residues per mole. There is

no suggestion^of a trend in the number of residues of a single

amino-acid and the complexity of the animal, as hypothesized

for methionine (Gracy et a_l., 1970).

Several investigators have attempted to relate homologies

in the structure of aldolases through a variety of methods

(Rutter, 1964; 1965; Rutter e_t al., 1963; Rutter et al., 1963;

Penhoet et al., 1967; Rutter et al., 1968; Anderson et al.,

1969; Idehara et al., 1969; Koida et al., 1969). Molecular

weight, effect of chelators, specificity of substrates (FDP/

F1P ratio), electrophoretic mobilities, terminal amino acids,

and tryptic and cyanogen bromide peptide patterns, all have

very little quantitative significance from a phylogenetic

51

standpoint. Other homologies between different aldolases

have been attempted through the use of amino-acid sequences

of the active site peptide (Guha et al., 1971; Lai and Oshima,

1971), but the results have yielded limited evolutionary in-

formation (vida infra).

Until the complete primary sequence of amino-acid in

proteins, including aldolase, are completed, other means of

quantitative comparisons must be sought. Slobin (1970) com-

pared the Metzger difference index (Metzger et al., 1968) to

the number of sequence changes in various cytochromes c. He

reported that a value of less than 7 or less than 9 resulted

in sequence likenesses of 90$ and 80%, respectively. Metzger's

data ( I968) indicates that unrelated proteins seldom yield a

value of less than 15.

This study has utilized three statistical procedures,

the correlation coefficient (Steele and Torrie, 1962), the

deviation function (Harris et al., 1969) , and the difference

index (Metzger et al. . 1968), to compare amino-acid compositions

of numerous aldolases (Appendix A). TableVHC displays the

amino-acid composition of Ascaris aldolase with aldolases from

16 other sources, using the three comparative procedures. The

correlation between the independent comparisons is very high.

The deviation function (DF) and difference index (Dl) have a

correlation coefficient (CC) of O.98, while the correlation

value for DF-CC and DI-CC comparisons are greater than 0.95.

DE and DI can be interconverted from the relationship DI=1.65 (DF).

TABLE VIII

COMPARISONS OF DEVIATION FUNCTION, DIFFERENCE INDEX, AND CORRELATION COEFFICIENT OF ALDOLASE AMINO-ACID COMPOSITIONS

52

Ascaris Compared With: DF DI CC

Ox

H

3.02 4.50 .976

Human 4.33 7.35 .942

Pig 2.97 4.42 .973

Rat 2.85 4.43 .965

Rabbit A 2.82 4.45 .976

Rabbit B 4.18 6.92 .951

Rabbit C 4.90 8.30 .933

Chicken 3 i 28 5.07 . 966

Snake 5.18 8.73 .923

Frog 3.63 6.12 .960

Codfish 3.37 6 .12 .966

Sturgeon 4.00 7.03 .950

Shark 4.67 7.28 .944

Lobster 4.92 8.82 .926

Spinach 4.46 7.38 .953 Yeast 5.25 8.97 .911

53

Extending this study, 136 pairs of aldolases from 17

sources were compared, employing the deviation function (Table

IX). The average DF values between mammals were used as a

standard value (1.32). The DF values of the other classes

of animals were then compared to this standard value. By

using this method it is readily seen that as the phylogenetic

scale is descended from mammals the DF values gradually in-

crease. This is evidence of increasing dissimilarities in

the amino-acid compositions. These DF differences were then

plotted against the geologic age in which the classes evolved

(except for mammals, where the modern orders of evolution were

used) (Fig. 12). There is a direct correlation between the

increasing DF and increasing evolutionary time. The animal

classes fall in exceptionally close order to the classical

phylogenetic sequence. To confirm this relationship, the

sequence changes in cytochromes c were also plotted against

time. The evolutionary pathway of this respiratory protein

has been quantitatively established (Dickerson et al., 1971;

Margoliash et al., I965). Thus, cytochrome c is an excellent,

reference to compare the findings of aldolase phylogeny.

Correlation between the DF of aldolase and sequence changes

in cytochrome c was exceptionally high, a value greater than

O.98 (Table X). There is little correlation (O.63) between

the interclass comparisons, ie., birds-fish, fish-reptiles, etc.

Homologous aldolase isozymes from muscle tissue of various

species have more similarity than heterologous isozymes (muscle,

. 54,

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55

Fig. 12, Plot of DF of aldolases and changes in cyto-chromes c against the time of evolution of each Class (except for mammals).

56

600

• • SEQUENCE CHANGES IN CYTO C

10 15 20 25

500

ac 400 < LU >~

Li— o co 300

200

100

ARTHROPODS

PISCES • ©

Q O AMPHIBIANS

ASCARIS REPTILES

PRESENT

Q© AVES

MAMMALS

1

DEVIATION FUNCTION ©—©

OF ALDOLASE

57

TABLE X

CORRELATION AND COMPARISON OF DF OF ALDOLASE WITH SEQUENCE CHANGES IN CYTOCHROMES C

Comparison DF Cyto. c Changes

Mammals - Mammals 1-32 4.75

Chicken 2.12 9.9

Snake 3.99 20.3

Frog 2 .66 12.1

Codfish 2.961 i 17.7 (tuna) CC=>0.98

Sturgeon 3.18 ' 3.40

Shark 4.07,

Ascaris 2.88

Lobster 4.29 24.6 (insect)

58.

liver, brain) from the same animal (rabbit) as previously

reported (Marquart, 1971). Furthermore, muscle and liver

isozymes are more closely related than muscle and brain or

liver and brain.

These results may be compared with reports from the

laboratory of Horecker (Guha et al., 1971), who has compared

the active site amino-acid sequences of rabbit, frog, codfish,

sturgeon, and lobster. The number of sequence alterations

per 28 amino-acid-peptide is as follows:

Comparison Sequence Changes

Rabbit-frog 4 -codfish 5 -sturgeon 4 -lobster 5 -liver 5

Frog-codfish 4 -sturgeon 2 -lobster 4

Codfish-sturgeon 4 -lobster 6

Sturgeon-lobster 6

Using this method, it is difficult to reveal any descent

through the classes studied. Applying the studies of Ingram

(1961) and Zuckerkandl and Pauling (1962) to the relationship

of myglobin-hemoglobin series, these substitutions give the

appearance that lobster muscle aldolase is more closely re-

lated to rabbit than to either of the lower vertebrates,

codfish or sturgeon.

59

The active sites do show that muscle aldolases are homo-

logous and exhibit extreme conservativism. For instance, the

sequence around the Schiff-base forming lysine (15), residues

9-22, is identical for all aldolases determined. Likewise,

cysteine is always located at residue 25 and methionine at

position 18, even in the extreme case when just one methionine

is present in the total peptide chain (Ting et al., 1971)•

Thus, the active site is not likely to be indicative of the

sequence changes that are capable of occurring in less crit-

ical regions of the proteins.

Molecular paleontology can be a very valuable tool. Data

obtained from hemoglobins (Ingram, 196l) and cytochromes c

(Dickerson et al. 1971) have greatly contributed in substan-

tiation of previously established geological records, and

have also provided data during periods in which fossil records

are absent or incomplete. This is readily seen in the present

study. From the DF of aldolases it appears that Ascaris

evolved approximately 300 million years ago (Fig. 12), with

the advent of terrestrial vertebrates. This is in contrast

with the presently accepted time of 600 million years for

free-living roundworms. It is conceivable that Ascaris

evolved later than the free-living roundworms since this

parasite has "adapted" to become totally dependent upon its

host. Also, Ascaris has been protected from selection pres-

sures by living in the well-regulated environment of the

intestine. It would be interesting to see if the cytochrome

60

c sequence supports this theory, since this protein has re-

cently been purified from Ascaris (Hill et al., 1971).

Further, fossil records of Ascaris are absent since it is

soft-bodied and is found within the body of its host. Cau-

tion and discretion must be used to prevent overextending

the information gained thus far. More animals and more

proteins must be examined before a molecular phylogenetic

tree can be confirmed.

From this quantitative study of aldolase the question

arose pertaining to the relationship of Class I and Class

II aldolases. Because of the diverse catalytic and molec-

ular differences, Rutter (1964; 1965) proposed that the two

classes were analogous, evolving from unique genes. These

2 classes of aldolase then are considered as examples of

convergent evolution. At the time of these proposals (1964)*

Class I aldolases were reported to be trimers with subunit

molecular weights of 50,000 (Deal et al., 1963; Kowalsky

and Boyer, I960; Stellwagen and Schachman, 1962) and Class

II aldolases dimers, subunit molecular weight, 35*000 (Rutter,

1964; 1965). New developments have since taken place. The

subunit molecular weights are essentially identical, 40,000

(Harris et al., 1969; Kawahara and Tanford, I966). The

deviation values (Table IX) for yeast and animals fall be-

tween 5.14 and 6.91. This displays a relative degree of

similarity, since the DF for unrelated proteins is seldom less

61

than 8.8 (Metzger et al., 1968)'. Harris et al. (1969) show

DF values for rabbit A-yeast comparisons to be 6.25, rabbit

A~cytochrome c, 13.6, and rabbit A-chymotrypsin, 12.1.

By employing Rutter's original model of aldolase evolu-

tion, the apparent 'homology' of Class I and II aldolases

may be explained.

Class II

Ancestral Gene

Gene

Dupli-cation

Metal Requirement Conserved Mutations

Properties Retained

T (Class I

Useless Non-functional Gene

> Free

Mutations

More Purposeful Gene

'Creative Event'

Most organisms possess a great deal of gene redundancy.

When duplicity is present, metabolically meaningful variants

tend to develop (Rutter, 1964). A duplicate gene thus may

undergo unconserved mutations, free from selective pressures.

This "useless" gene may develop into a functional genome.

Finally this "created" genome may then express itself and aid

in the physiological needs of the organism. Thus, from a

common ancestral gene, becoming progressively more divergent,

• 5 K Value obtained by converting DI to DF.

62

Class I aldolases may have mutated significantly to develop

a more purposeful catalytic mechanism. The length of the

genome (MW of the subunits) and amino-acid composition (DF)

have been conserved.

This study, as well as previous ones, foims the basis

for a comparative study of aldolase. There is a vast number

of invertebrates yet unstudied that would provide pertinent

and valuable information. A great deal of insight might be

gained from examining all of the pre-adult stages of Ascaris.

Aldolases from several animals in the same class would con-

firm a clear picture of aldolase phylogeny. Such studies

would yield a confirmed molecular phylogenetic tree in a

relatively short period of time (in comparison to sequencing).

It is cleaij then, that this study of aldolase may just be a

springboard for a wide variety of further comparisons.

SUMMARY

1. Fructose-1,6-diphosphate aldolase has been purified

157-fold from "the intestinal roundworm, Ascaris suum. with '

a final specific activity of 10.2 units per min per mg.

2. The enzyme was adjudged to be homogeneous by the

criteria of analytical ultracentrifugation, zone electro-

phoresis, and rechromatography.

3. Concomitant with the homogeneity studies, the follow-

ing physical parameters were established: S^Q w = 7.98 x

10 ^ sec; D 2 Q = 4.43 x 10 ? cm2per sec; Stokes' radius

o =46.4 A; f/fQ = 1.29.

4. Sedimentation velocity, sedimentation equilibrium,

and analytical gel filtration yield a molecular weight of

158,000 + 4000 for the native enzyme. The enzyme dissociates

in 6 M guanidinium-Cl and SDS to four identical subunits of

molecular weight 41,000.

5. The apparent Michaelis constants determined for

FDP and F1P were J x 10"5 and 3 x 10~2, respectively. The

catalytic ratio (FDP/F1P) was 42. The enzyme was unaffected

by EDTA or potassium ions.

6. Comparison of the tryptic peptide maps indicate a

high degree of homology between the mammalian (rabbit muscle)

and Ascaris, aldolases. This indicates that the enzyme has

retained its structural properties during its evolution.

63

64

7. The aldolase amino acid compositions from 17 sources

have been compared by the deviation function, difference

index, and correlation coefficient. A proposal of a phyloge-

netic study of aldolases utilizing these quantitative methods

of comparing proteins from their amino acid compositions

is discussed.

65

APPENDIX A

Description of Procedures Used for Comparisons of Amino-Acid Compositions

Deviation Function (DF) = L ~ ̂ n^-}2 *

Difference Index (Dl) = £|"̂ n ~ ̂ n| * ^0

Correlation Coefficient (CC) = £(XY) - (£x) (^Y)/n

AI^X)2 - iix)2/n-(W2-<.(^/n

All amino-acid compositions were normalized to 100,000

MW. X and Y represent mole fractions of corresponding amino-

acids from the pair of compositions compared. Tryptophan was

not included in calulations.

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