Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.
We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology*; D002784:Cholesterol / Q000097:blood / Q000378:metabolism*; D002791:Cholesterol, Dietary / Q000494:pharmacology*; D002792:Cholestyramine Resin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006706:Homeostasis; D007700:Kinetics; D007810:Lanosterol / Q000378:metabolism*; D008148:Lovastatin / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride / Q000494:pharmacology
D000924:Anticholesteremic Agents; D002791:Cholesterol, Dietary; D002792:Cholestyramine Resin; D007810:Lanosterol; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride; D002784:Cholesterol; D008148:Lovastatin; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase