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- (F1) and three NADPH-dependent (F2, F3 and F4)] by DEAE-cellulose column chromatography. By several procedures, F2 was purified over 1000-fold and F4 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 F2 and F4, 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 F2 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 18.104.22.168). The F4 enzyme, on the other hand, preferentially acted on C24 3-ketosteroids.
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