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Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

ABSTRACT: Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.

SUBMITTER: Hart GJ

PROVIDER: S-EPMC1164692 | biostudies-other | 1977 May

REPOSITORIES: biostudies-other

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Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

Project description:The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.

| S-EPMC1186151 | biostudies-other

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

Project description:The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

| S-EPMC1158276 | biostudies-other

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

Project description:The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

| S-EPMC1144564 | biostudies-other

Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram.

Project description:Stoicheiometric amounts of [14C]disulfiram react rapidly with sheep liver cytoplasmic aldehyde dehydrogenase to give loss of catalytic activity and incorporation of the expected amount of radioactivity. In a subsequent slower reaction the label is lost from the enzyme without re-emergence of enzymic activity. The results imply that in vivo disulfiram may act as an oxidation-reduction catalyst for the inactivation of aldehyde dehydrogenase.

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Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase.

Project description:Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD(+) and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD(+) and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD(+), estimates of 2x10(5) litre.mol(-1).s(-1) and 2s(-1) were obtained for k(+1) and k(-1) respectively. Although k(+3) could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s(-1), k(+2) and k(-2) could not be determined uniquely, but could be related by the equation: (k(-2)+k(+3))/k(+2) =50x10(-6)mol.litre(-1). No significant isotope effect was observed when [1-(2)H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k(-2)+k(+3))/k(+2)=2.3x10 (-3)mol.litre(-1) and k(+3) is 23s(-1). When [1,2,2,2-(2)H]acetaldehyde was used, no isotope effect was observed on k(+3), but there was a significant effect on k(+2) and k(-2). A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.

| S-EPMC1183679 | biostudies-other

The removal of cytosolic-type aldehyde dehydrogenase from preparations of sheep liver mitochondrial aldehyde dehydrogenase and the unusual properties of the purified mitochondrial enzyme in assays.

Project description:The pI approximately 5.2 isoenzymes of mitochondrial aldehyde dehydrogenase were separated from the other isoenzymes by pH-gradient chromatography on DEAE-Sephacel. The pI approximately 5.2 material is immunologically identical with cytosolic aldehyde dehydrogenase. It also shows sensitivity to 20 microM-disulfiram and insensitivity to 4M-urea in assays. These and other criteria seem to establish that the material is identical with the cytosolic enzyme. Mitochondrial enzyme that had been purified to remove pI approximately 5.2 isoenzymes shows concentration-dependent lag phases in assays. These effects are possibly due to the slow establishment of equilibrium between tetramer and either dimers or monomers, with the dissociated species being intrinsically more active than the tetramer.

| S-EPMC1144409 | biostudies-other

Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase.

Project description:Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

| S-EPMC1158499 | biostudies-other

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

Project description:The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

| S-EPMC1183996 | biostudies-other

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

Project description:The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.

| S-EPMC1186043 | biostudies-other

Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents.

Project description:Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2'-dithiodipyridine and 4,4'-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4'-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2'-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5'-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

| S-EPMC1138814 | biostudies-other

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