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Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes.

1984; Elsevier BV; Volume: 259; Issue: 3 Linguagem: Inglês

10.1016/s0021-9258(17)43459-4

1083-351X

Yuri Aoyama, Yuichi Yoshida, Ryosuke Sato,

Pharmacogenetics and Drug Metabolism

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-45ol4n~, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14a-trimethyl-5a-cholesta-8,24-dien-38-01) to a sterol metabolite in the presence of NADPH and molecular oxygen.This conversion did not occur anaerobically or when either P -4 6 0 1 4 ~~, the reductase, or NADPH was omitted from the system.In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns.Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol(4,4-dimethyl-5cr-cholesta-8,24-dien-3@-01) indicated that the metabolite was 4,4-dimethyl-5a-cholesta-8,14,24-trien-3~-01.Upon aerobic incubation of microsomes from semianaerobkally grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol.This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-45014D~.It is concluded that in yeast microsomes lanosterol is 14a-demethylated by a P-4501aDM-containing monooxygenase system to give rise to 4,4-dimethyl-5a-cholesta-8,14,24-trien-3@-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.' The abbreviations used are: DLPC, dilauroylphosphatidylcholine; GC-MS, gas chromatography