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, 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.
Kedzie
K M
KM
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Balfour
C A
CA
Escobar
G Y
GY
Grimm
S W
SW
He
Y A
YA
Pepperl
D J
DJ
Regan
J W
JW
Stevens
J C
JC
Halpert
J R
JR
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
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