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Biomedical subjects

M J Hardman

Publications and source records attributed to M J Hardman.

At least 19 recordsLinked to original sources
PubMed1992

Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose-fructose oxidoreductase from Zymomonas mobilis.

The reduction of gluconolactone by glucose-fructose oxidoreductase containing tightly bound NADPH (enzyme-NADPH) is biphasic in nucleotide fluorescence. The initial rapid decrease, which represents quenching of the fluorescence by bound lactone, is followed by a slower decrease which corresponds to the change in absorbance. At low glucose concentrations, the oxidation of glucose by enzyme-NADP+ involves a single first-order process with similar rate constants in fluorescence and absorbance. At higher glucose concentrations, the apparent first-order rate constants for the fluorescence change are less than those for the absorbance change. This is consistent with a mechanism in which the fluorescence change occurs during the lactone dissociation step, which is slower than the hydrogen transfer step during which the absorbance change occurs. The rate constant for gluconolactone dissociation is 360 +/- 10 s-1 and this step is therefore rate-determining for the overall reaction. Reduction of fructose by enzyme-NADPH is first order with a limiting rate constant of at least 2000 s-1.

Carbohydrate Dehydrogenases↗
PubMed1992

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.

Adenosine Triphosphate↗
PubMed1991

The importance of alcohol dehydrogenase in regulation of ethanol metabolism in rat liver cells.

We used titration with the inhibitors tetramethylene sulphoxide and isobutyramide to assess quantitatively the importance of alcohol dehydrogenase in regulation of ethanol oxidation in rat hepatocytes. In hepatocytes isolated from starved rats the apparent Flux Control Coefficient (calculated assuming a single-substrate irreversible reaction with non-competitive inhibition) of alcohol dehydrogenase is 0.3-0.5. Adjustment of this coefficient to allow for alcohol dehydrogenase being a two-substrate reversible enzyme increases the value by 1.3-1.4-fold. The final value of the Flux Control Coefficient of 0.5-0.7 indicates that alcohol dehydrogenase is a major rate-determining enzyme, but that other factors also have a regulatory role. In hepatocytes from fed rats the Flux Control Coefficient for alcohol dehydrogenase decreases with increasing acetaldehyde concentration. This suggests that, as acetaldehyde concentrations rise, control of the pathway shifts from alcohol dehydrogenase to other enzymes, particularly aldehyde dehydrogenase. There is not a single rate-determining step for the ethanol metabolism pathway and control is shared among several steps.

Acetaldehyde↗
PubMed1991

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.

Animals↗
PubMed1988

The kinetics of glucose-fructose oxidoreductase from Zymomonas mobilis.

Glucose-fructose oxidoreductase operates by a classic ping-pong mechanism with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol. The Km values for these substrates were determined. The values of kcat are 200 s-1 and 0.8 s-1 for the forward and reverse directions respectively. The overall catalytic process consists of two half-reactions with alternate reduction of NADP+ and oxidation of NADPH tightly bound to the enzyme. Reduction of enzyme-NADP+ by glucose and oxidation of enzyme-NADPH by gluconolactone involve single first-order processes. The values of the rate constants at saturating substrate are 2100 s-1 and 8 s-1 respectively; deuterium isotope effects indicate that these are for the hydrogen transfer step. Oxidation of enzyme-NADPH by fructose is first order with a limiting rate constant of at least 430 s-1. The reaction of enzyme-NADP+ with sorbitol is biphasic, with rate constants for both phases less than 1 s-1. This behaviour is explained by a mechanism in which the slow cyclisation of the acyclic form of fructose follows its dissociation from the enzyme. The rate-determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme-bound NADP+ in the reverse direction.

Carbohydrate Dehydrogenases↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Kinetics of activation of L-lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate and by metal ions.

The lactate dehydrogenase from Streptococcus faecalis is activated either by fructose 1,6-bisphosphate or by divalent cations such as Mn2+ or Co2+. With both types of activator, a lag is observed before attainment of the steady state rate of pyruvate reduction if the activator is added to the enzyme at the same time as the substrates. This lag can be largely abolished by preincubation of enzyme with activator before mixing with substrates. For fructose 1,6-bisphosphate (Fru(1,6)P2) as the activator, the rate constant for the lag phase showed a linear dependence on activator concentration but was independent of enzyme concentration. This suggests that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme which leads to increased activity, without association of enzyme subunits or dimers. With Co2+ as activator, the rate constant for the lag phase showed a hyperbolic dependence on Co2+ concentration and was also dependent on enzyme concentration. This suggests that activation by Co2+, in contrast to that by Fru(1,6)P2, involves association of enzyme dimers, followed by ligand binding.

Cobalt↗
PubMed1987

Evidence that the slow conformation change controlling NADH release from the enzyme is rate-limiting during the oxidation of propionaldehyde by aldehyde dehydrogenase.

The displacement of NADH from the aldehyde dehydrogenase X NADH complex by NAD+ was followed at pH 7.0, and the data were fitted by a non-linear least-squares iterative procedure. At pH 7.0 the decay constants for the dissociation of NADH from aldehyde dehydrogenase X NADH complexes (1.62 +/- 0.09 s-1 and 0.25 +/- 0.004 s-1) were similar to the values previously determined by MacGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462] at pH 7.6, and apparent differences between these values and those reported by Dickinson [(1985) Biochem. J. 225, 159-165] are resolved. Experiments at low concentrations of propionaldehyde show that isomerization of a binary E X NADH complex is part of the normal catalytic mechanism of the enzyme. Evidence is presented that the active-site concentration of aldehyde dehydrogenase is halved when enzyme is pre-diluted to low concentrations before addition of NAD+ and substrate. The consequences of this for the reported values of kcat. are discussed. A general mechanism for the aldehyde dehydrogenase-catalysed oxidation of propionaldehyde which accounts for the published kinetic data, at concentrations of aldehyde which bind only at the active site, is presented.

Aldehyde Dehydrogenase↗
PubMed1985

Carboxymethylated liver alcohol dehydrogenase: pH dependence of hydride transfer during ethanol oxidation.

The rate constant for the hydride transfer step during oxidation of ethanol by carboxymethylated alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) is dependent on a group with pKa 7.5. This pKa is higher than that for the native enzyme. A mechanism is proposed to account for the ionisation and the protein fluorescence change which occur during formation of the carboxymethylated enzyme-NAD+-ethanol complex.

Alcohol Dehydrogenase↗
PubMed1985

Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate.

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6-bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose-1,6-bisphosphate concentration, with a second-order rate constant of 2.0 X 10(4) M-1 s-1, but were independent of enzyme concentration. Binding of fructose 1,6-bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second-order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction. The results suggest that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.

Chemical Phenomena↗
PubMed1985

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.

Alcoholism↗
PubMed1983

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.

Amino Acids↗
PubMed1983

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.

Alcohol Dehydrogenase↗