scholarly journals Pathways of reducing equivalents in hepatocytes from rats. Estimation of cytosolic fluxes by means of 3H-labelled substrates for either A- or B-specific dehydrogenases

1987 ◽  
Vol 243 (3) ◽  
pp. 625-630 ◽  
Author(s):  
C Vind ◽  
A Hunding ◽  
N Grunnet
Keyword(s):  
Experimental Data ◽  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Least Squares ◽  
Oxidation Rate ◽  
Isolated Hepatocytes ◽  
Glycerol Phosphate ◽  
Hydrogen Atoms ◽  
Best Fit ◽  
Glycerol Phosphate Dehydrogenase

The metabolism of [2-3H]lactate and [2-3H]glycerol was studied in isolated hepatocytes from fed rats. In order to estimate the rate of equilibrium between the 4A and 4B hydrogen atoms of NADH, we compared the flow of 3H from [2-3H]lactate and [2-3H]glycerol, the oxidations of which are catalysed by A- and B-type dehydrogenases, respectively. Hepatocytes were incubated with lactate, glycerol and ethanol and tracer amounts of [2-3H]lactate or [2-3H]glycerol and the labelling rates of lactate, ethanol, glucose and glycerol phosphate were determined. The data were used to calculate the oxidation rate of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase, triosephosphate dehydrogenase and glycerol phosphate dehydrogenase. The rates were calculated by obtaining the best fit of a model to the experimental data by using a least-squares procedure. The results support our model and suggest that the fluxes through various dehydrogenases are sufficient to equilibrate the 4A and 4B hydrogen atoms of cytosolic NADH. The validity of the metabolic models used was evaluated by comparison of rates of NADH oxidation catalysed by cytosolic dehydrogenases as calculated by two different models.

scholarly journals The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose

1984 ◽  
Vol 222 (2) ◽  
pp. 437-446 ◽  
Author(s):  
C Vind ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Maximal Activity ◽  
Isolated Hepatocytes ◽  
Single Compartment ◽  
Dehydrogenase Reaction ◽  
Order Of Magnitude ◽  
Rate Limiting

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.

scholarly journals Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

1989 ◽  
Vol 86 (17) ◽  
pp. 6464-6468 ◽  
Author(s):  
D K Srivastava ◽  
P Smolen ◽  
G F Betts ◽  
T Fukushima ◽  
H O Spivey ◽  
...  
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Transfer Mechanism ◽  
Rabbit Muscle ◽  
Direct Transfer ◽  
Glycerol Phosphate ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Random Diffusion ◽  
Glycerol Phosphate Dehydrogenase

Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of alpha-GDH and LDH.

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