Further studies of the action of disulfiram and 2,2′-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase

Abstract

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2'-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2'-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.

Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent

“…We found 2 mol of dimethyldithiocarbamate bound per mol of tetrameric ALDH and an equal amount of free dimethyldithiocarbamate, proving that the bound reagent was not displaced from the enzyme. These results are similar to those found with the sheep enzyme (Kitson, 1982) if proper reaction conditions are employed.…”

“…It was demonstrated with human (Vallari & Pietruszko, 1982) and then with sheep (Kitson, 1982) cytosolic ALDH that the bound diethyldithiocarbamate could be displaced by a second cysteine residue, forming an internal disulphide bond in the enzyme. This reaction did not occur with the horse mitochondrial enzyme under the conditions of the experiment.…”

“…The total radioactivity associated with protein was essentially equal to that found for the [35S]dimethyl compound. This observation suggests that the mode of binding to tetramethylthiuram disulphide to the horse liver mitochondrial enzyme is through a mixed disulphide bond, analogous to the mechanism proposed by Neims et al (1966), and shown to occur by Kitson (1982). The number of sites modified was calculated from the specific radioactivity associated with the modified enzyme, assuming the following inactivation scheme:…”

“…Thus it appears that disulfiram may not be an active-site-directed reagent. Further, with sheep (Dickinson et al, 1981;Kitson, 1982) and human (Vallari & Pietruszko, 1982) liver enzyme, maximal, but not total, inhibition is achieved when only two mol of disulfiram have been bound per mol of tetrameric enzyme.…”

Distribution and Properties of Aldehyde Dehydrogenase in Regions of Rat Brain

“…Why complete inhibition of ALDH was not obtained is not known. Conceivably, disulfiram is not an active site-directed agent as originally reported, but only reduces the specific activity of the enzyme, as has been suggested to occur with liver enzyme (Sanny and Weiner, 1977;Dickinson et al, 1981;Kitson, 1982). More detailed work is required to determine if the full complement of brain ALDH is modified by disulfiram or if, after drug treatment, the enzyme exists as a mixture of inactive and active forms.…”