title: Molecular basis for a functionally unique cytochrome P450IIB1 variant.
abstract: Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.
authors.affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
authors.forename: K M
authors.initials: KM
authors.lastname: Kedzie
authors.forename: C A
authors.initials: CA
authors.lastname: Balfour
authors.forename: G Y
authors.initials: GY
authors.lastname: Escobar
authors.forename: S W
authors.initials: SW
authors.lastname: Grimm
authors.forename: Y A
authors.initials: YA
authors.lastname: He
authors.forename: D J
authors.initials: DJ
authors.lastname: Pepperl
authors.forename: J W
authors.initials: JW
authors.lastname: Regan
authors.forename: J C
authors.initials: JC
authors.lastname: Stevens
authors.forename: J R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000483:Alleles; D000818:Animals; D003001:Cloning, Molecular; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D014644:Genetic Variation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011917:Rats, Inbred Lew; D011919:Rats, Inbred Strains; D011920:Rats, Inbred WF; D011921:Rats, Inbred WKY; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D039181:Steroid 16-alpha-Hydroxylase; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D039181:Steroid 16-alpha-Hydroxylase; D010634:Phenobarbital