Partial characterization of alcohol dehydrogenase activity in purified rat Leydig cells.
Ethanol metabolism to acetaldehyde by NAD+-dependent alcohol dehydrogenase (ADH) activity reduces, in part, androgen secretion by rat Leydig cells. ADH in Leydig cells is proposed to decrease the NAD+/NADH ratio and thereby inhibit NAD+-dependent delta 5-3 beta hydroxysteroid dehydrogenase-isomerase activity and increase NADH-dependent 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase activity. Although the reciprocal changes in these steroidogenic enzyme activities by ethanol are attributed to ADH activity, there is very little information about this enzyme in purified Leydig cells. The present studies examined specific characteristics of this enzyme in metrizamide-gradient purified Leydig cells. ADH activity was linear with respect to protein concentration and incubation time. The activity was concentrated in the soluble fraction, and the most effective cofactor was NAD+. The apparent Km for ethanol was 0.50 mM, and the Vmax was 53 nmol NADH/10 min/mg protein. When Leydig cell cytosol was incubated with a fixed ethanol concentration (50 mM) and increasing NAD+ and the data were plotted according to Lineweaver-Burk, a biphasic curve was observed with apparent Km's of 0.032 and 0.17 mM. The optimum pH for the enzyme was 8.2, and the enzyme was inhibited in a dose-dependent manner by 4-methylpyrazole. These studies further characterize ADH activity in purified Leydig cells and demonstrate that this enzyme exhibits many characteristics similar to the more widely studied liver enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)
D000079:Acetaldehyde / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000032:analysis*; D000818:Animals; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology; D000077604:Fomepizole; D006863:Hydrogen-Ion Concentration; D007985:Leydig Cells / Q000201:enzymology*; D008297:Male; D009243:NAD / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D011720:Pyrazoles; D009243:NAD; D000431:Ethanol; D000077604:Fomepizole; D000426:Alcohol Dehydrogenase; D000079:Acetaldehyde