Molecular basis for a functionally unique cytochrome P450IIB1 variant

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, Wis...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 266; no. 33; pp. 22515 - 22521
Main Authors:	Kedzie, K M, Balfour, C A, Escobar, G Y, Grimm, S W, He, Y A, Pepperl, D J, Regan, J W, Stevens, J C, Halpert, J R
Format:	Journal Article
Language:	English
Published:	Bethesda, MD American Society for Biochemistry and Molecular Biology 25-11-1991
Subjects:	alanine Alleles Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Cloning, Molecular Cytochrome P-450 CYP2B1 Cytochrome P-450 Enzyme System - genetics Cytochrome P-450 Enzyme System - isolation & purification Cytochrome P-450 Enzyme System - metabolism cytochrome P450IIB1 DNA - genetics DNA - isolation & purification endoplasmic reticulum Fundamental and applied biological sciences. Psychology Genetic Variation Hemoproteins Kinetics Liver - enzymology Male Metalloproteins Microsomes, Liver - drug effects Microsomes, Liver - enzymology Oxidoreductases - genetics Oxidoreductases - isolation & purification Oxidoreductases - metabolism Phenobarbital - pharmacology Plasmids Proteins Rats Rats, Inbred Lew Rats, Inbred Strains Rats, Inbred WF Rats, Inbred WKY Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism RNA - genetics RNA - isolation & purification Sequence Homology, Nucleic Acid Species Specificity Steroid 16-alpha-Hydroxylase Substrate Specificity Hemoprotein Purification Rat Enzyme Cytochrome P450 Liver Genetic variant Rodentia Inactivation Characterization Vertebrata Mammalia Substrate specificity
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Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0021-9258 1083-351X
DOI:	10.1016/s0021-9258(18)54602-0