Studies on NADPH-dependent chloral hydrate reducing enzymes in rat liver cytosol

Abstract

Chloral hydrate, a sedative hypnotic and also a major metabolite of trichloroethylene in higher animals, is reduced to trichloroethanol by liver extracts. The reducing activity in rat liver cytosol could be separated into four fractions [one NADH- (F 1) and three NADPH-dependent (F 2, F 3 and F 4)] by DEAE-cellulose column chromatography. By several procedures, F 2 was purified over 1000-fold and F 4 was purified over 600-fold from liver cytosol. As judged from polyacrylamide gel electrophoresis performed with and without the addition of sodium dodecylsulfate, the final preparations were essentially homogeneous. They differed in molecular weight, mobility on polyacrylamide gel electrophoresis, pH optimum, substrate specificity, and sensitivity to inhibitors. The molecular weights were estimated to be 36,000 and 32,500 for F 2 and F 4, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The estimation of molecular weights by thin-layer gel chromatography indicated that the enzymes were monomers. An examination of over thirty substrates revealed that both enzymes catalyzed the reduction of long-chain aliphatic, alicyclic and aromatic aldehydes as well as halogenated acetaldehyde. The F 2 enzyme acted on d-glucuronate, indicating that it was identical to the aldehyde reductase recently reported by other workers ( l-gulonate: NADP + 1-oxidoreductase EC 1.1.1.19). The F 4 enzyme, on the other hand, preferentially acted on C 24 3-ketosteroids.