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 1991-11, Vol.266 (33), p.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: Article
Language:English
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
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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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)54602-0