Yeast Cytochrome P-450 Catalyzing Lanosterol 14 Alpha-Demethylation. II. Lanosterol Metabolism by Purified P-450(14)DM and by Intact Microsomes

J Biol Chem. 1984 Feb 10;259(3):1661-6.

Abstract

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, 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-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically 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-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aerobiosis
  • Anaerobiosis
  • Cytochrome P-450 Enzyme System / metabolism*
  • Gas Chromatography-Mass Spectrometry
  • Immunodiffusion
  • Kinetics
  • Lanosterol / metabolism*
  • Microsomes / metabolism*
  • Saccharomyces cerevisiae / metabolism*

Substances

  • Lanosterol
  • Cytochrome P-450 Enzyme System