Michael J. Hardman

Kinetic studies on the thiol protease from Actinidia chinensis

The plant thiol proteases, papain, ficin and bromelain show a high degree of homology in the amino acid sequence near the active residues [ 1,2] and kinetic studies of papain and ficin indicate that the specificity and mechanism of action are similar [2,3]. A further thiol protease, actinidin [4], from the berries of Actinidia Chinensis (Chinese gooseberries) was first characterised by Arcus [4] and later studied by McDowall [5] . We have purified the enzyme from unripe berries to constant specific activity as a sulphenyl thiosulphate derivative and carried out kinetic studies to compare the behaviour of this enzyme with that of the other thiol proteases. The specificity of the enzyme towards small substrates and inhibitors, and its activity towards N-c&BZ-lysine p-nitrophenyl ester are similar to those of ficin and papain.

The specificity of actinidin and its relationship to the structure of the enzyme

The kinetic parameters kcat, Km and kcat/Km, have been determined for the actinidin-catalysed hydrolyses of N-substituted amino acid esters and amides and compared to the corresponding values for papain (EC 3.4.22.2). Substrates with aromatic N-substituents have lower kcat/Km values for actinidin (EC 3.4.22.14); the difference is much smaller for substrates with aliphatic substituents. The lower kcat/Km values for actinidin generally correspond to higher Km values suggesting that the strength of substrate binding differs between the two enzymes. This difference is explained in terms of the differences in the substrate binding sites found in X-ray crystallographic studies.

The assessment of viability in isolated rat hepatocytes

Isolated rat hepatocytes are used in many metabolic studies, but the viability of these cell preparations is often not adequately established. The present study shows that ATP content is a more reliable index of metabolic viability than trypan blue exclusion. At some of the low trypan blue exclusion levels quoted in the literature, a high percentage of cell preparations is likely to be nonviable by the criterion of ATP content. We suggest that ATP content measured on initial cell preparations and at the end of all incubation procedures is essential for establishing cell viability for metabolic studies on isolated hepatocytes.

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.

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.

The effect of premixing on the oxidation of ethanol by liver alcohol dehydrogenase

Abstract Contrary to our predictions, premixing of enzyme with NAD has little effect on the transient phase of the oxidation of ethanol by liver alcohol dehydrogenase. This observation has led us to re-examine the theory of premixing effects and to consider the consequences of this theory for the reduction of aldehydes by alcohol dehydrogenase.

The complement component C1s catalysed hydrolysis of peptide 4-nitroanilide substrates

The kinetic parameter kcat/Km has been determined for the hydrolysis of peptide 4-nitroanilides, catalysed by complement component C1s. Substrates based on the C-terminal sequence of human C4a (Leu-Gln-Arg) were synthesised. Replacement of the glutamine residue by glycine or serine increased kcat/Km. Substitution of valine for the leucine residue increased kcat/Km, while substitution of glycine or lysine for the leucine residue decreased kcat/Km slightly. D-Val-Ser-Arg 4-nitroanilide is the most reactive 4-nitroanilide substrate towards C1s, so far. These results are discussed in relation to the amino acid sequences near the bonds cleaved by C1s in C4, C2 and C1 inhibitor.

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.

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.

Regulation of Rates of Ethanol Metabolism and Liver [NAD+]/[NADH] Ratio

Factors that control the rate of alcohol metabolism in mammals have been the subject of debate for many years (for detailed reviews, see Crow, 1985; Crow and Hardman, 1989). There have been two main theories as to the major rate limitation on the ethanol metabolic pathway. The first theory was that the rate at which NADH (generated in the alcohol and aldehyde dehydrogenase reactions) could be reoxidised to NAD+ was limiting (Hawkins and Kalant, 1972; Khanna and Israel, 1980; Thurman et al, 1989). This theory arose from the observation that the ratio of free [NAD+]/[NADH] in liver cytosol decreased during ethanol metabolism. It was assumed either that the liver ran out of NAD+, because the rate of reoxidation of NADH was limiting, and this lack of NAD+ then limited the rate of the alcohol dehydrogenase (ADH) reaction (Khanna and Israel, 1980) or that NADH accumulated and caused product inhibition of ADH (Thurman et al, 1989). The second theory was that the amount of ADH present in the liver was the main rate-determining factor for the pathway (Crow et al, 1977; Cornell et al, 1979; Braggins and Crow, 1981; Cornell, 1983; Bosron et al, 1983). This theory arose in part from observations that the activity of liver ADH measured in vitro was only slightly more than necessary to explain rates of ethanol metabolism observed in vivo (Crow et al, 1977; Cornell et al, 1979; Braggins and Crow, 1981). ADH was not present ‘in excess’ as had sometimes been claimed (Hawkins and Kalant, 1972; Kalant et al, 1975). It was also observed that variations in ADH activity induced by castration (Rachamin et al, 1980; Mezey et al, 1980; Cicero et al, 1980, 1982), starvation (Braggins and Crow, 1981; Lumeng et al, 1979, 1980) or stress (Mezey et al, 1979) were associated with corresponding changes in rates of ethanol metabolism.