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 Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria

1978 ◽  
Vol 175 (3) ◽  
pp. 899-908 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Maximum Rate ◽  
Liver Mitochondria ◽  
Enzyme Concentration ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Control Value ◽  
Sheep Liver ◽  
Substrate Activation ◽  
Variable Substrate

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.

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 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 dissociation of glucose oxidase by sodium n-dodecyl sulphate

1982 ◽  
Vol 203 (1) ◽  
pp. 285-291 ◽  
Author(s):  
M N Jones ◽  
P Manley ◽  
A Wilkinson
Keyword(s):  
Glucose Oxidase ◽  
Specific Binding ◽  
Enzymic Activity ◽  
Sedimentation Equilibrium ◽  
Prolonged Incubation ◽  
Rate Analysis ◽  
Subunit Molecular Weight ◽  
A Subunit ◽  
Glycine Methyl Ester ◽  
Cationic Residues

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.

scholarly journals Purification of aldehyde dehydrogenase from rat liver mitochondria by α-cyanocinnamate affinity chromatography

1989 ◽  
Vol 259 (1) ◽  
pp. 105-110 ◽  
Author(s):  
R C Poole ◽  
A P Halestrap
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Aldehyde Dehydrogenase ◽  
Minor Component ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Column ◽  
A Subunit ◽  
A Minor ◽  
Related Compounds

1. alpha-Cyano-4-hydroxycinnamate was coupled to Sepharose CL-4B activated with 1,2:3,4-bisepoxybutane. 2. The low-Km rat liver mitochondrial aldehyde dehydrogenase was specifically bound to this affinity medium, and could subsequently be eluted with alpha-cyano-4-hydroxycinnamate. 3. The enzyme purified in this manner had a subunit molecular mass of 55 kDa and a pI of approx. 6.5. A minor component of approx. 57 kDa was also present and had a significantly higher pI value; this may be the precursor for aldehyde dehydrogenase. 4. alpha-Cyanocinnamate and some related compounds were found to be uncompetitive inhibitors of the enzyme. 5. No cytosolic aldehyde dehydrogenase was bound to the affinity column, but a protein from a rat liver post-mitochondrial supernatant with a molecular mass of approx. 25 kDa was bound, and could be eluted subsequently with alpha-cyano-4-hydroxycinnamate.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

1969 ◽  
Vol 115 (4) ◽  
pp. 639-643 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Equilibrium Sedimentation ◽  
Purification And Properties

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.

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.

Modification of thiol groups in cytoplasmic aldehyde dehydrogenase

Alcohol ◽  
1985 ◽  
Vol 2 (1) ◽  
pp. 97-101 ◽  
Author(s):  
Trevor M. Kitson ◽  
Kerry M. Loomes
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Thiol Groups
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