Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase.
The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.
D000818:Animals; D002784:Cholesterol / Q000096:biosynthesis*; D003579:Cytochrome Reductases / Q000302:isolation & purification; D003580:Cytochromes / Q000302:isolation & purification; D007202:Indicators and Reagents; D007700:Kinetics; D007810:Lanosterol / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013261:Sterols / Q000138:chemical synthesis; D013379:Substrate Specificity
D003580:Cytochromes; D007202:Indicators and Reagents; D013261:Sterols; D007810:Lanosterol; D002784:Cholesterol; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase; D003579:Cytochrome Reductases