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

1989 ◽  
Vol 261 (1) ◽  
pp. 281-284 ◽  
Author(s):  
K M Loomes ◽  
T M Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Related Compound ◽  
Physiological Responses ◽  
Enzymic Activity ◽  
Cysteine Residues ◽  
Mitochondrial Enzyme ◽  
Step Process ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

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.

scholarly journals The separation of sheep liver cytoplasmic and mitochondrial aldehyde dehydrogenases by isoelectric focusing, and observations on the purity of preparations of the cytoplasmic enzyme, and their sensitivity towards inhibition by disulfiram

1979 ◽  
Vol 179 (3) ◽  
pp. 709-712 ◽  
Author(s):  
F M Dickinson ◽  
S Berrieman
Keyword(s):  
Isoelectric Focusing ◽  
Aldehyde Dehydrogenase ◽  
Liver Enzymes ◽  
Mitochondrial Enzyme ◽  
Selective Precipitation ◽  
Aldehyde Dehydrogenases ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver ◽  
Ph Range

Preparations of sheep liver cytoplasmic aldehyde dehydrogenase obtained by published methods were found by analytical isoelectric focusing in the pH range 5–8 to contain 5–10% by weight of the mitochondrial aldehyde dehydrogenase. Under the conditions used the pI of the cytoplasmic enzyme is 6.2 and that of the mitochondrial enzyme 6.6. The mitochondrial enzyme can be removed from the preparation by selective precipitation of the cytoplasmic enzyme with (NH4)2SO4. Kinetic experiments and inhibition experiments with disulfiram show that the properties of the two sheep liver enzymes are so different that the presence of 10% mitochondrial enzyme in preparations of the cytoplasmic enzyme can introduce serious errors into results. Our results suggest that the presence of 10 microM-disulfiram in assays may completely inactivate the pure cytoplasmic enzyme. This result is in contrast with a previous report [kitson (1978) Biochem. U. 175, 83–90].

scholarly journals Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver

1978 ◽  
Vol 175 (2) ◽  
pp. 753-756 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Acetic Anhydride ◽  
Mitochondrial Enzyme ◽  
Oxidation Reactions ◽  
Aldehyde Dehydrogenases ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver ◽  
Michaelis Constants ◽  
Butyric Anhydride ◽  
Partial Reversal

In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V′max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V′max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively.

scholarly journals The activation of aldehyde dehydrogenase by diethylstilboestrol and 2,2′-dithiodipyridine

1982 ◽  
Vol 207 (1) ◽  
pp. 81-89 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Enzyme Activity ◽  
Aldehyde Dehydrogenase ◽  
Human Liver ◽  
Common Property ◽  
Rabbit Liver ◽  
Aldehyde Dehydrogenases ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver ◽  
Low Concentrations ◽  
Liver Aldehyde Dehydrogenase

1. The activation of sheep liver cytoplasmic aldehyde dehydrogenase by diethylstilboestrol and by 2,2′-dithiodipyridine is described. The effects of the two modifiers are very similar with respect to variation with acetaldehyde concentration, pH and temperature. Thus the degree of activation is maximal when the enzyme is assayed at approx. 1 mM-acetaldehyde, is greater at 25 degrees C than at 37 degrees C, and is greater at pH 7.4 than at pH 9.75. With low concentrations of acetaldehyde both modifiers decrease the enzyme activity. 2. Diethylstilboestrol affects the sheep liver cytoplasmic enzyme in a very similar way to that previously described for a rabbit liver cytoplasmic enzyme. Preliminary experiments show that the same is true for a preparation of human liver aldehyde dehydrogenase. It is proposed that sensitivity to diethylstilboestrol (and steroids) is a common property of all mammalian cytoplasmic aldehyde dehydrogenases.

scholarly journals 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

1984 ◽  
Vol 224 (1) ◽  
pp. 163-169
Author(s):  
S Allanson ◽  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
The Other ◽  
Ph Gradient ◽  
Mitochondrial Enzyme ◽  
Sheep Liver ◽  
Species Being ◽  
Gradient Chromatography ◽  
Lag Phases

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.

scholarly journals Some properties of aldehyde dehydrogenase from sheep liver mitochondria

1977 ◽  
Vol 163 (2) ◽  
pp. 261-267 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Liver Mitochondria ◽  
Enzymic Activity ◽  
Sedimentation Equilibrium ◽  
Thiol Groups ◽  
Control Value ◽  
Sheep Liver ◽  
Spectrophotometric Titrations ◽  
A Subunit ◽  
Subunit Size

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.

scholarly journals Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram

1983 ◽  
Vol 213 (2) ◽  
pp. 551-554 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Catalytic Activity ◽  
Aldehyde Dehydrogenase ◽  
Enzymic Activity ◽  
Sheep Liver ◽  
Oxidation Reduction

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.

scholarly journals The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram

1981 ◽  
Vol 199 (3) ◽  
pp. 573-579 ◽  
Author(s):  
F M Dickinson ◽  
G J Hart ◽  
T M Kitson
Keyword(s):  
Ion Exchange ◽  
Aldehyde Dehydrogenase ◽  
Active Sites ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Gradient ◽  
Original Activity ◽  
Covalent Interaction ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH.

scholarly journals The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver

1976 ◽  
Vol 155 (2) ◽  
pp. 445-448 ◽  
Author(s):  
T M. Kitson
Keyword(s):  
Mitochondrial Enzyme ◽  
Aldehyde Dehydrogenases ◽  
Covalent Interaction ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver ◽  
Structural Analogues

The effect of some close structural analogues of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. Several thiuram disulphides are equally potent inhibitors of the cytoplasmic enzyme, but in all cases the mitochondrial enzyme is much less strongly affected. Tetramethylthiuram monosulphide decreases the activity of the cytoplasmic enzyme in a process apparently involving a covalent interaction.

scholarly journals Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase

1985 ◽  
Vol 225 (1) ◽  
pp. 159-165 ◽  
Author(s):  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Dissociation Reaction ◽  
Quenching Effect ◽  
Sheep Liver ◽  
Association Reaction ◽  
Hydrolysis Of

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.

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