Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism.

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.

The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes.

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.

Effects of ethanol treatment and castration on liver alcohol dehydrogenase activity.

Induction of alcohol dehydrogenase (ADH) activity by chronic ethanol treatment and castration has previously been reported to occur in Sprague-Dawley rats. In the present study, no induction was found following chronic ethanol treatment and only a low level of induction was found with castration. However the activity of ADH was high in control animals compared with those used in other studies. The activity of ADH in control animals was not decreased by testosterone administration, which has been shown to reverse induction of the enzyme produced by chronic ethanol treatment or castration in other studies. It is concluded that the male Sprague-Dawley rat is not necessarily a suitable animal for the study of ADH induction by chronic ethanol treatment and that further unknown factors must be identified before the regulation of ADH activity in vivo is fully understood.

Increased malate dehydrogenase activity in blood from non-drinking alcoholics.

Since cytosolic malate dehydrogenase has been shown to play a role in the regulation of liver cytosolic [NAD+]/[NADH] redox state during ethanol metabolism, it is possible that differences in this enzyme could cause differences in response to ethanol. The present study demonstrates that the isozyme pattern of this cytosolic enzyme in whole blood samples is the same as that in liver and that the pattern does not differ in alcoholic and control subjects. A marginally significant elevation of activity of malate dehydrogenase in blood from alcoholic subjects is reported. Further studies are needed to confirm this latter finding and to assess fully its possible significance.

Human liver cytosolic malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism.

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 +/- 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (microM) of the kinetic parameters were obtained at pH 7.4 and 37 degrees C: Ka, 17; Kia, 3.6; Kb, 51; Kib, 68; Kp, 770; Kip, 10,700; Kq, 42; Kiq, 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD+, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 mumol/min/g wet wt of liver for oxalacetate reduction and 11.2 mumol/min/g liver for malate oxidation at pH 7.4 and 37 degrees C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic NAD+/NADH redox state during ethanol metabolism in human liver.

Kinetics of malate dehydrogenase and control of rates of ethanol metabolism in rats.

The theory that the rate of ethanol oxidation is governed by rates of NADH reoxidation is based in part on the observation that the ratio of free cytosolic [NADH]/[NAD+] increases during ethanol metabolism. However, it has recently been suggested that the amount of alcohol dehydrogenase governs rates of ethanol metabolism, which then leaves the change in cytosolic redox state unexplained. In this paper the kinetic parameters for rat liver malate dehydrogenase, determined at 37 degrees C and pH 7.4, are used to provide an explanation for the change in cytosolic redox state that is compatible with rate control by alcohol dehydrogenase.

Factors influencing rates of ethanol oxidation in isolated rat hepatocytes.

The stimulation of ethanol oxidation by fructose which has frequently been observed in isolated hepatocytes was found to occur only in unsupplemented cells. In the presence of other substrates (lactate, pyruvate) which accelerate ethanol oxidation, fructose had no additional effect. Acceleration of ethanol oxidation by fructose was not directly related to the ATP demand created by fructose. The effects of fructose on ethanol oxidation rates were not due to changes in acetaldehyde concentration. In cells from fed animals, acetaldehyde concentrations rose as high as 200 microM in some incubations, and therefore became a significant factor limiting ethanol oxidation rates. In hepatocytes isolated from starved rats incubated with pyruvate, where acetaldehyde concentrations were very low, (1-2 microM) it was possible to assess the effect of changes in [lactate]/[pyruvate] (and hence free cytosolic NADH) on rates of ethanol oxidation. The results showed that the increase in free cytosolic [NADH] usually found during ethanol oxidation in vivo would inhibit rates of ethanol clearance by a maximum of 20%.

A two-site model for the esterase and dehydrogenase activities of sheep liver aldehyde dehydrogenase.

Although aldehyde dehydrogenase (ALDH) from sheep liver cytosol has a broad specificity, it will not oxidize the aldehyde group of glyoxylic acid which is in fact an inhibitor of the enzyme. The inhibition pattern is non-linear but competitive at high propionaldehyde concentrations (2-20 mM); however, a simple non-competitive pattern is observed at low (less than 100 microM) propionaldehyde concentrations (Ki = 1.6 mM). The esterase activity was unaffected by glyoxylic acid in the absence of NAD+ but a simple competitive inhibition pattern (Ki = 2.5 mM) was observed with respect to 4-nitrophenyl acetate in the presence of NAD+. The data require a two-site model in which ester and aldehyde binding sites are distinct but with a second propionaldehyde molecule, and glyoxylic acid, binding at or near the ester binding site. Consistent with this model is the fact that chloral hydrate was a non-competitive inhibitor of the esterase activity in the presence of NAD+ but a competitive inhibitor in its absence. The enzyme exhibited hysteretic behavior governed by the protonated form of an ionizable group with an apparent pKa of 7.55.

The effects of high ethanol doses on rates of ethanol oxidation in rats. A reassessment of factors controlling rates of ethanol oxidation in vivo.

1. Ethanol was oxidised more slowly by rats which were given an ethanol dose of 5.1 g/kg than by rats which were given an ethanol dose of 1.4 g/kg. 2. A positive correlation was found between [lactate]/[pyruvate] ratios and rates of ethanol oxidation. 3. Acetaldehyde concentrations varied widely between rats, but in some cases were high enough to influence rates of ethanol oxidation. 4. Liver alcohol dehydrogenase levels were just sufficient to account for ethanol oxidation rates observed in vivo. 5. Pre-administration of a large ethanol dose (6.5 g/kg) did not alter mean [lactate]/[pyruvate] ratios or ethanol oxidation rates during metabolism of test doses of 2.5 g/kg. 6. Injection of pyruvate did not increase rates of ethanol oxidation. 7. The results do not support suggestions that a high-Km ethanol oxidising system plays an important role in vivo, that increased rates of ethanol oxidation can be induced by large, acute ethanol doses or that the rate of NADH reoxidation controls rates of ethanol metabolism. 8. The results support other evidence which has indicated that the level of alcohol dehydrogenase is the major factor limiting rates of ethanol oxidation in vivo.

A reinvestigation of the purity, isoelectric points and some kinetic properties of the aldehyde dehydrogenases from sheep liver.

1. Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the mitochondrial enzyme by isoelectric focusing. 2. Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pI = 5.22 +/- 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pI = 5.22 and five further bands of pI 5.48 +/- 0.09, 5.56 +/- 0.07, 5.65 +/- 0.06, 5.70 +/- 0.03 and 5.76 +/- 0.02. Possible origins of the isoenzymes are discussed. 3. Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram-modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group. 4. The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4-nitrophenoxide ion during the hydrolysis of 4-nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 +/- 1 s-1 and the steady-state rate constant was 0.2 s-1, values similar to those previously reported for the cytoplasmic enzyme. 5. The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s-1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady-state mechanism is discussed.

Re-activation by glutamate or aspartate of amino-oxyacetate-inhibited aspartate aminotransferase in vitro and in isolated hepatocytes.

Experiments with isolated rat hepatocytes and with cell extracts indicate, in contrast with previous reports, that pyruvate does not block or reverse the inhibition of aspartate aminotransferase (EC 2.6.1.1) by amino-oxyacetate. That inhibition, however, is partially overcome by glutamate or aspartate either in cell extracts or in whole cells incubated with substrate combinations that cause accumulation of those amino acids.