Redox States and Phosphorylation Potentials Bob Harris raharris@iupui.edu October 5, 2010

Redox States and Phosphorylation Potentials

Bob Harris

raharris@iupui.edu

October 5, 2010

Redox States

• NAD+/NADH cytoplasm• NAD+/NADH mitochondria• NADP+/NADPH cytoplasm• NADP+/NADPH mitochondria

Measuring the NAD+ redox state

• Usually expressed as ratio of [NAD+]/[NADH]• Total NAD+ divided by total NADH?• Free NAD+ divided by free NADH?• Make any difference if we use total values or

free values?

Cytosolic NAD+/NADH ratios based on total

(free and bound) NAD+ and NADH in rat liver

State NAD+ NADH NAD+/NADH

mol/g

Fed 0.76 0.14 5.4

Starved0.82 0.16 5.1

Cytosolic NAD+/NADH ratios based on free concentrations

State NAD+/NADH

Fed 725

Starved 528

Calculating the NAD+ redox state

Free values obtained by measuring metabolites of an equilibrium enzyme

Lactate + NAD+ pyruvate + NADH + H+

Keq = [pyruvate][NADH][H+] / [lactate][NAD+]

[NAD+]/[NADH] = [pyruvate][H+] / [lactate] x 1/Keq

Equilibrium constants

• Equilbrium constants: for A B; Keq = [B]/[A]

• Mass action ratios: MAR = [B]/[A]

• Equilibrium enzymes:high activity; Keq = MAR

• Nonequilibrium enzymes:low activity; Keq= MAR

A B C D E F

Cytoplasmic free NAD+/NADH

Lactate dehydrogenase catalyzes equilibrium reaction:Lactate + NAD+ pyruvate + NADH + H+

Keq = [pyruvate][NADH][H+] / [lactate][NAD+]

[NAD+]/[NADH] = [pyruvate][H+] / [lactate] x Keq

Set pH = 7.0 and incorporate into Keq

K’eq = [pyruvate][NADH]/ [lactate][NAD+]

[NAD+]/[NADH] = [pyruvate]/[lactate] x 1/K’eq

Example of calculation

Freeze clamp liver of fed wild type mice:Lactate: 1.09 0.09 mol/g wet wtPyruvate: 0.12 0.01 mol/g wet wt

K’eq @ pH 7.0 = 1.11 x 10-4

[NAD+]/[NADH] = [pyruvate]/[lactate] x 1/K’eq

[NAD+]/[NADH] = [0.120]/[1.09] x 1/1.11 x 10-4

[NAD+]/[NADH] = 991

Effect of ethanol on liver cytosolic NAD+/NADH ratio

Ethanol + NAD+ acetaldehyde + NADH + H+

Expect NADH drive pyruvate to lactate via:

Pyruvate + NADH +H+ Lactate + NAD+

Expect decrease in NAD+/NADH ratio

Effect of ethanol on liver cytosolic NAD/NADH ratio

Treatment NAD/NADH

Control 719

Ethanol (2 millimoles) 132*

*Five minutes after injection of ethanol.

Equilibrium enzymes used for calculations of free ratios

Mitochondrial free NAD+/NADH:-hydroxybutyrate dehydrogenase-hydroxybutyrate + NAD+ acetoacetate +

NADH + H+

K’eq @ pH 7.0 = 4.93 x 10-2

Glutamate dehydrogenaseGlutamate + NAD+ yields -ketoglutarate +

NADH + NH4+ K’eq @ pH 7.0 = 3.87 x 10-3 mM

Effect of starvation on liver mitochondrial NAD+ redox state

State NAD+/NADH NAD+/NADH (Free)* (Total)

Fed 7.3 2.2Starved 4.7 ND

*Calculated from concentrations of components of the glutamate dehydrogenase reaction.

Effect of ethanol on liver mitochondrial NAD/NADH ratio

Ethanol + NAD+ acetaldehyde + NADH + H+

Acetaldehyde + NAD+ acetate + NADH + H+

Expect NADH will drive -ketoglutarate to glutamate via:

-Ketoglutarate + NADH +NH4+ glutamate +

NAD+

Expect decrease in mitochondrial NAD+/NADH ratio

Effect of ethanol on liver mitochondrial NAD+/NADH ratio

Treatment NAD+/NADH

Control 7.7

Ethanol (2 millimoles) 2.7*

*Five minutes after injection of ethanol.

Equilibrium enzymes used for calculations of free ratios

Cytoplasmic free NADP+/NADPH6-phosphogluconate dehydrogenase:6-phosphogluconate + NADP+ ribulose 5-

phosphate + NADPH + H+ + CO2

Isocitrate dehydrogenase:Isocitrate + NADP+ -ketoglutarate + NADPH +

CO2

Malic enzyme:

Malate + NADP+ pyruvate + NADPH + H+ + CO2

Keq for NADP+ coupled enzymes

6-phosphogluconate dehydrogenase

1.17 M

Isocitrate dehydrogenase

1.72 x 10-1 M

Malic enzyme

3.44 x 10-2 M

Reactions catalyzed by NADP+ coupled enzymes produce CO2

CO2 concentration does not vary significantly under conditions that are normally studied.

Rather than measure, usually assumed to be 1.16 mM.

Caution: CO2 concentration is affected by changes in pH.

Typical values of cytoplasmic NADP+/NADPH

State NADP+/NADPH NADPH/NADP+

Fed 0.009 110

Starved 0.006 175

NADP+/NADPH ratio important

• Sets the ratio of GSH/GSSG in cytoplasm because of equilibrium enzyme reaction catalyzed by glutathione reductase

NADPH + H+ +GSSG 2 GSH + NADP+

• Driven far to the right because of very high NADPH/NADP+ ratio.

• Important in both cytoplasm and mitochondrial matrix space

NAD+/NADH ratio important for many reasons

• High cytoplasmic NAD/NADH ratio favors oxidation of substrates.

• Low cytoplasmic NAD/NADH results in low pyruvate and low oxaloacetate which inhibits glucose synthesis.

• Free NAD+ is activator of SIRT1 • Free NADH is activator of the PDKs• Both serve as both substrates and allosteric effectors

for many enzyme systems.

Phosphorylation potential

• Defined as [ATP]/[ADP][Pi]• Comes from:∆G = ∆Gº - RTln[ATP]/[ADP][Pi]• Two ways of determining

– From measurements of total ATP, ADP, and Pi (not accurate because total [ADP] >>free [ADP])

– From concentrations of metabolites of equilibrium enzymes (much more accurate)

Calculation of phosphorylation potentials

[ATP]/[ADP][Pi] = [NAD+]/[NADH] x [glyceraldehyde-3-P]/[3-phosphoglycerate] x KGAPDH x KPGK

Derivation

Glyceraldehyde-3-P + NAD+ + Pi yields 1,3- bis-Phosphoglycerate + NADH + H+

1,3-Phosphoglycerate + ADP yields 3-Phosphoglycerate + ATP

Sum: Glyceraldehyde-3-P + NAD+ + Pi + ADP yields 3-phosphoglycerate + ATP + NADH

KGAPDH x K3-PGK = [ATP]/[ADP][Pi] x [NADH]/[NAD+] x 3-[phosphoglycerate]/ [glyceraldehyde-3-P]

[ATP]/[ADP][Pi] = [NAD+]/[NADH] x [glyceraldehyde-3-P]/[3-phosphoglycerate] x KGAPDH x K3-PGK

Calculation of phosphorylation potentials

[ATP]/[ADP][Pi] = [NAD+]/[NADH][H+] x [glyceraldehyde-3-P]/[3-phosphoglycerate] x KGAPDH x K3-PGK

Obtain [NAD+]/[NADH] from [pyruvate]/[lactate] and KLDH

[ATP]/[ADP][Pi] = [pyruvate]/[lactate] x [glyceraldehyde-3-P]/[3-phosphoglycerate] x {KGAPDH x K3-PGK}/KLDH

Calculation of phosphorylation potentials

Obtain [glyceraldehyde-3-P] from [dihydroxyacetone-P] and the Keq (22) for triose phosphate isomerase

glyceraldehyde-3-P dihydroxyacetone-P

Keq = 22 = [dihydroxyacetone-P]/ [glyceraldehyde-3-P]

[glyceraldehyde-3-P] = [dihydroxyacetone-P]/22

Calculation of phosphorylation potentials

[ATP]/[ADP][Pi] = [pyruvate]/[lactate] x [dihydroxyacetone phosphate]/22 x 1/[3-phosphoglycerate] x {KGAPDH x K3-PGK}/KLDH

{KGAPDH x K3-PGK}/KLDH = 1.65 x 107 M-1

Typical metabolite values for freeze clamped rat liver

Metabolite mol/g wet wt

Lactate 1.36

Pyruvate 0.258

3-Phosphglycerate 0.387

Dihydroxyacetone P 0.043

ATP 3.38

ADP 1.32

AMP 0.294

Pi 4.76

Calculation of phosphorylation potentials

ATP ADP ATP/ADPxPi*

mol/g wet wt M-1

Total 3.38 1.32 531

Free 16,300

*[Pi] taken to be 4.8 mol/g

Calculation of free [ADP]

Free cytosolic [ADP] =

[ATP]/{[Pi] x phosphorylation potential}

Calculation of phosphorylation potentials

ATP ADP ATP/ADPxPi*

mol/g M-1

Total 3.38 1.32 531

Free 3.38 0.046 16,300

*[Pi] taken to be 4.8 mol/g; water content taken to be 0.8 grams per gram wet weight tissue.

Calculation of free [AMP]

From the equilibrium constant (1.05) for reaction catalyzed by myokinase:

ATP + AMP 2 ADP

Free cytosolic [AMP] =

{[free cytosolic ADP]2 x KMK}/[measured ATP]

Comparison of total measured [AMP] and calculated free [AMP]

ADP AMPmol/g wet wt

Total 1.32 0.294Free cytosolic 0.046 0.0007*

*0.7 nmoles/g wet weight!

Important points about adenine nucleotides

• Free [AMP] is much lower than total [AMP]- Important because [AMP] activates AMPK and functions as positive or negative allosteric effector for many enzymes.

• Free [ADP] is much lower than the total [ADP]– Important because [ADP] determines respiration

rate of mitochondria• Decrease in [ATP] results in increase in [AMP]

because of equilibrium reaction catalyzed by myokinase: 2 ADP ATP + AMP

Effect of fasting, exercise, hypoglycemia, high fat diet, and diabetes on liver adenine nucleotides

Berglund et al. JCI 119:2412–2422 (2009)

Hems and Brosnan. Effect of ischemia on content of metabolites in rat liver and kidney

Biochem J 1970; 120:105-111Well-fed rats

IschemiaATP ADP AMP AMP/ATP

(sec) (mol/g wet wt)

0 2.7 1.3 0.26 0.09

60 1.6 1.8 0.85 0.53

48-starved rats

IschemiaATP ADP AMP AMP/ATP

(sec) (mol/g wet wt)

0 1.7 2.0 0.64 0.37

60 0.9 1.7 1.65 1.83

Greenbaum et al. Hepatic metabolites and …. in animals of different dietary and hormonal status.

Arch. Biochem. Biophys. 1971; 143: 617-663

Metabolic

State ATP ADP AMP AMP/ATP

(mol/g wet wt)

Well-fed 1.9 0.91 0.23 0.12

Starved 1.7 1.0 0.31 0.18

Schewenke et al. Mitochondrial and cytosolic AT/ADP ratios in rat liver in vivo

Biochem J 1981; 200: 405-408

Metabolic

State ATP ADP AMP AMP/ATP

(mol/g dry wt)

Well-fed 3.3 0.86 0.16 0.05

Starved 2.7 0.82 0.18 0.07

Perhaps mice are not just small rats?

Our measurements on fed and fasted mice

Measurement Fed Fasted

mol/g wet wt

ATP 3.0 0.2 3.2 0.2

ADP 0.89 0.07 0.85 0.07

AMP 0.28 0.04 0.24 0.03

Why difference between our data and the data of Burgess et al.?

• Freeze clamping has to be done rapidly to preserve phosphorylation state of the adenine nucleotides.

• Burgess et al. Approximately 20 seconds.• Our study: Less than 8 seconds.

Faupel et al. The problem of tissue sampling from experimental animals….. ABB 1972; 148: 509-522

Faupel et al. The problem of tissue sampling from experimen-tal animals…. ABB 1972; 148: 509-522

Freeze clamp protocol

1. Three people who can work together are required. One to manage stop watch; one strong person to handle freeze clamps; one person with good hands to kill mouse by cervical dislocation, open mouse with a single cut with scissors, tear out liver, and place on freeze clamp.

2. Practice until steps 4, 5, and 6 can be completed by team in less than 8 seconds. Discard any samples not clamped in less than 8 seconds.

3. Handle mice on several days prior to the experiment in the room in which the mice will be killed. Transport the mice to the killing room one at a time. 

4. Person 1: start stopwatch at time of cervical dislocation; stop at time liver clamped.

5. Person 2: kill mouse by cervical dislocation with large pair of scissors; open mouse with a single cut with same scissors; tear out liver by hand; place liver on freeze clamps.

6. Person 3: clamp tissue with as much force as possible with liquid-nitrogen cooled clamps.

7. Clean the area and instruments before bringing the next mouse to the killing room.  (Mice are stressed by the odor of blood).  

Effect of fasting, exercise, hypoglycemia, high fat diet, and diabetes on liver adenine nucleotides

Berglund et al. JCI 119:2412–2422 (2009)

Our measurements on chow and high fat fed mice

Measurement Chow High Fat Dietmol/g wet wtATP 3.0 0.2 2.7 0.2ADP 0.89 0.07 1.14 0.05*AMP 0.28 0.04 0.42 0.02* *P < 0.05

Best way to measure ATP, ADP, and AMP?

• Enzyme-coupled assays?• HPLC?

Direct comparison of enzymatic and HPLC method for nucleotide quantification

Measurement Enzymatic HPLCmol/g wet wtATP 3.0 0.2 2.7 0.2ADP 0.89 0.07 1.8 0.1*AMP 0.28 0.04 0.6 0.1*

*P < 0.05

References

Faupel, RP, Seitz, HJ, Tarnowski, W., Thiermann, V, Weiss, C. The problem of tissue sampling from experimental animals with respect to freezing technique, anoxia, stress and narcosis. ABB (1972) 148: 509-522.

Veech, RL, Guynn, R, Veloso, D. The time-course of the effects of ethanol on the redox and phosphorylation states of rat liver. Biochem. J. (1972) 127, 387-397.

Veech, RL, Lawson, JWR, Cornell, NW, Krebs, HA. Cytosolic phosphorylation potential. JBC (1979) 254: 6538-6547.

Berglund,ED, Lee-Young, RS, Lustig, DG, Lynes, SE, Donahue,P, Camacho, RC., Meredith, ME., Magnuson, MA, Charron, MJ, Wasserman, DH. Hepatic energy state is regulated by glucagon receptor signaling in mice. JCI (2009) 119: 2412-2422.

Importance of AMP/ATP ratio

• AMP is a positive allosteric effector of:– Glycogen phosphorylase (glycogenolysis)– PFK1 (glycolysis) – AMP kinase (glycolysis; Fatty acid

oxidation; inhibit gluconeogenesis)

• ATP is a negative allosteric effector of:– Pyruvate kinase (glycolysis)

Resveratrol

High fat diet

SIRT1

SREBP1c PGC1

FOXFAS

Ethanol

PPAR

Lipoic acid

Shong et al. The effect of feeding high fat

diet on NQO1 expression. In preparation

Park et al. Lipoic Acid Decreases Lipogenesis via AMPK-Dependent and –Independent Pathways. Hepatology 2008; 48:1477-1486

• Lipoic acid in diet – reduced hepatic steatosis.– increased AMPK activity– Inhibited SREBP1c expression– Increased capacity for fatty acid oxidation

Park et al. Lipoic Acid Decreases Hepatic Lipogenesis Through AMPK-Dependent and AMPK-IndependentPathways HEPATOLOGY, Vol. 48, No. 5, 2008

Park et al. Lipoic Acid Decreases Hepatic Lipogenesis Through AMPK-Dependent and AMPK-IndependentPathways HEPATOLOGY, Vol. 48, No. 5, 2008

NQO1• NQO1 = “old yellow enzyme” = DT

diaphorase (D = DPN (NAD+) ; T = TPN (NADP+); NAD(P)H:quinone acceptor oxidoreductase; cytoplasmic enzyme

• NAD(P)H + H+ + electron acceptor (EA)

yields NAD(P)+ + H2EA– Important point: catalyzes 2 electron

transfer as opposed to one electron transfer that could produce O2•

-Lapachone

• Best known synthetic substrate for NQO1– Lowest Km; highest Vmax

• NAD(P)H + H+ + Lap yields NAD(P)+ + LapH2

• Approved in some countries as anti-cancer agent

Effect of -Lapachone in fat-fed miceHwang et al. Stimulation of NADH oxidation ameliorates obesity and related

phenotypes in mice. Diabetes 58: 965-974, 2009

• Increased hepatic NAD+/NADH ratio. – Increased AMPK activity– Increased PGC1 and SIRT1– Decreased acetyl-CoA carboxylase

activity– Increased fatty acid oxidation– Ameliorated adiposity, glucose intolerance,

dyslipidemia, and fatty liver in mice fed high fat diet

Shin et al. -Lapachone alleviates alcoholic

fatty liver disease in rats. In preparation

• In alcohol-fed mice, -Lapachone– reduced hepatic steatosis – Increased hepatic fatty acid oxidizing

capacity– Increases NAD/NADH ratio– Increased AMPK activity

High fat diet

Resveratrol (PDK KO???)SIRT1

SREBP1c PGC1

FOXFAS

Ethanol

NAD+

Smile

ERR

PDK4

p53

PDK2

Phenotype of NQO1 knockout mice

• Decreased hepatic NAD/NADH ratio

• Reduces fasting blood levels of glucose in chow fed and high fat fed mice

• Reduces steatosis in high fat fed mice