title: Expression of rabbit cytochrome P-450IIE2 in yeast and stabilization of the enzyme by 4-methylpyrazole.
abstract: A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.
authors.affiliation: Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109.
authors.forename: S J
authors.initials: SJ
authors.lastname: Pernecky
authors.forename: T D
authors.initials: TD
authors.lastname: Porter
authors.forename: M J
authors.initials: MJ
authors.lastname: Coon
mesh_terms: D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D004247:DNA / Q000302:isolation & purification; D004795:Enzyme Stability / Q000187:drug effects; D000077604:Fomepizole; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011720:Pyrazoles / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility
chemical_list: C014059:CYC1 protein, S cerevisiae; D002793:Cholic Acids; D003574:Cytochrome c Group; D011720:Pyrazoles; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D000077604:Fomepizole; D045304:Cytochromes c; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D019826:Cholic Acid