Purification and Properties of Sheep‐Liver Aldehyde Dehydrogenases

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.

Section: Methodsmentioning

confidence: 99%

Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram

Kitson¹

1983

“…140nmol of NADH produced/min per mg in the standard assay), and performing the experiment immediately after enzyme isolation to avoid slight losses of activity on storage, the observed stoicheiometry did not exceed 2. This suggests that the enzyme, although composed of four subunits (MacGibbon et al, 1979), may have only two functional active sites. The same conclusion was reached by Takahashi & Weiner (1980) with respect to the pI-5 isoenzyme of horse liver.…”

Section: Resultsmentioning

confidence: 99%

“…The enzyme concentration was calculated for the tetrameric enzyme species based on mol.wt. 212000(MacGibbon et al, 1979) and A " ,= 1 1.3 at 280nm.…”

mentioning

confidence: 93%

“…Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH.In the past few years there have been several reports of various properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver (see e.g. Kitson, 1978;Hart & Dickinson, 1978;MacGibbon et al, 1979). Dickinson & Berrieman (1979) showed that their isolation procedure, which was based on that of Crow et al (1974), resulted in cytoplasmic enzyme preparations that were appreciably contaminated with the mitochondrial form.…”

mentioning

confidence: 99%

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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

Dickinson¹,

Hart²,

Kitson³

1981

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.

“…Cytoplasmic aldehyde dehydrogenase was purified from sheep liver as described previously (MacGibbon et al, 1979). Acetic anhydride from May and Baker (Dagenham, Essex, U.K.) was distilled before use, and 4nitrophenyl acetate from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.) was recrystallized from acetone.…”

Section: Methodsmentioning

confidence: 99%

Evidence that the cytoplasmic aldehyde dehydrogenase-catalysed oxidation of aldehydes involves a different active-site group from that which catalyses the hydrolysis of 4-nitrophenyl acetate

Motion

Buckley

Bennett

et al. 1988

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.