Transcript Document

Redox States and
Phosphorylation Potentials
Bob Harris
[email protected]
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
Fed
Starved
NAD+
NADH
mol/g
0.76
0.14
0.82
0.16
NAD+/NADH
5.4
5.1
Cytosolic NAD+/NADH ratios based
on free concentrations
State
Fed
Starved
NAD+/NADH
725
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 wt
Pyruvate: 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
Ethanol (2 millimoles)
*Five minutes after injection of ethanol.
719
132*
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 dehydrogenase
Glutamate + 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
Fed
Starved
NAD+/NADH
(Free)*
7.3
4.7
NAD+/NADH
(Total)
2.2
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+/NADPH
6-phosphogluconate dehydrogenase:
6-phosphogluconate + NADP+
ribulose 5phosphate + 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
Fed
Starved
NADP+/NADPH NADPH/NADP+
0.009
0.006
110
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 3phosphoglycerate + 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]/[3phosphoglycerate] x KGAPDH x K3-PGK
Calculation of phosphorylation
potentials
[ATP]/[ADP][Pi] = [NAD+]/[NADH][H+] x [glyceraldehyde3-P]/[3-phosphoglycerate] x KGAPDH x K3-PGK
Obtain [NAD+]/[NADH] from [pyruvate]/[lactate] and KLDH
[ATP]/[ADP][Pi] = [pyruvate]/[lactate] x [glyceraldehyde3-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/[3phosphoglycerate] 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
Total
Free
ATP
ADP
mol/g wet wt
3.38
1.32
*[Pi] taken to be 4.8 mol/g
ATP/ADPxPi*
M-1
531
16,300
Calculation of free [ADP]
Free cytosolic [ADP] =
[ATP]/{[Pi] x phosphorylation potential}
Calculation of phosphorylation
potentials
Total
Free
ATP
ADP
mol/g
3.38
1.32
3.38
0.046
ATP/ADPxPi*
M-1
531
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]
Total
Free cytosolic
ADP
AMP
mol/g wet wt
1.32
0.294
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; 120:105-111
Well-fed rats
Ischemia
ATP 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
Ischemia
ATP ADP AMP
(sec)
(mol/g wet wt)
0
1.7
2.0
0.64
60
0.9
1.7
1.65
AMP/ATP
0.37
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
Well-fed
ATP ADP AMP
(mol/g wet wt)
1.9
0.91 0.23
Starved
1.7
1.0
0.31
AMP/ATP
0.12
0.18
Schewenke et al. Mitochondrial and cytosolic
AT/ADP ratios in rat liver in vivo
Biochem J 1981; 200: 405-408
Metabolic
State
Well-fed
ATP ADP AMP
(mol/g dry wt)
3.3
0.86 0.16
Starved
2.7
0.82
0.18
AMP/ATP
0.05
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: 509522
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 liquidnitrogen 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
mol/g wet wt
ATP
3.0  0.2
ADP
0.89  0.07
AMP
0.28  0.04
*P < 0.05
High Fat Diet
2.7  0.2
1.14  0.05*
0.42  0.02*
Best way to measure ATP, ADP, and
AMP?
• Enzyme-coupled assays?
• HPLC?
Direct comparison of enzymatic and HPLC
method for nucleotide quantification
Measurement Enzymatic
mol/g wet wt
ATP
3.0  0.2
ADP
0.89  0.07
AMP
0.28  0.04
*P < 0.05
HPLC
2.7  0.2
1.8  0.1*
0.6  0.1*
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)
High fat diet
PPAR
Ethanol
SIRT1
Resveratrol
SREBP1c
PGC1
FAS
FOX
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-Independent
Pathways HEPATOLOGY, Vol. 48, No. 5, 2008
Park et al. Lipoic Acid Decreases Hepatic Lipogenesis
Through AMPK-Dependent and AMPK-Independent
Pathways 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 anticancer agent
Effect of -Lapachone in fat-fed mice
Hwang 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
NAD+
Ethanol
SIRT1
Resveratrol (PDK KO???)
p53
SREBP1c
FAS
PDK2
PGC1
FOX
Smile
ERR
PDK4
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