Molecular basis for a functionally unique cytochrome P450IIB1 variant

Karen M. Kedzie, Celia A. Balfour, Gina Y. Escobar, Scott W. Grimm, You Ai He, David J. Pepperl, John W Regan, Jeffrey C. Stevens, James R. Halpert

Research output: Contribution to journalArticle

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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 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-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 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-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 (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-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.

Original languageEnglish (US)
Pages (from-to)22515-22521
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number33
StatePublished - 1991

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Androstenedione
Cytochromes
Mixed Function Oxygenases
Rats
Liver Microsomes
Liver
7-Alkoxycoumarin O-Dealkylase
Inbred Strains Rats
Stereoselectivity
Hydroxylation
Amino Acid Substitution
Phenobarbital
Metabolites
Refractory materials
Sprague Dawley Rats
Testosterone
Substitution reactions
Complementary DNA
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kedzie, K. M., Balfour, C. A., Escobar, G. Y., Grimm, S. W., He, Y. A., Pepperl, D. J., ... Halpert, J. R. (1991). Molecular basis for a functionally unique cytochrome P450IIB1 variant. Journal of Biological Chemistry, 266(33), 22515-22521.

Molecular basis for a functionally unique cytochrome P450IIB1 variant. / Kedzie, Karen M.; Balfour, Celia A.; Escobar, Gina Y.; Grimm, Scott W.; He, You Ai; Pepperl, David J.; Regan, John W; Stevens, Jeffrey C.; Halpert, James R.

In: Journal of Biological Chemistry, Vol. 266, No. 33, 1991, p. 22515-22521.

Research output: Contribution to journalArticle

Kedzie, KM, Balfour, CA, Escobar, GY, Grimm, SW, He, YA, Pepperl, DJ, Regan, JW, Stevens, JC & Halpert, JR 1991, 'Molecular basis for a functionally unique cytochrome P450IIB1 variant', Journal of Biological Chemistry, vol. 266, no. 33, pp. 22515-22521.
Kedzie KM, Balfour CA, Escobar GY, Grimm SW, He YA, Pepperl DJ et al. Molecular basis for a functionally unique cytochrome P450IIB1 variant. Journal of Biological Chemistry. 1991;266(33):22515-22521.
Kedzie, Karen M. ; Balfour, Celia A. ; Escobar, Gina Y. ; Grimm, Scott W. ; He, You Ai ; Pepperl, David J. ; Regan, John W ; Stevens, Jeffrey C. ; Halpert, James R. / Molecular basis for a functionally unique cytochrome P450IIB1 variant. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 33. pp. 22515-22521.
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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 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-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 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-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 (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-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.",
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AU - Balfour, Celia A.

AU - Escobar, Gina Y.

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AU - He, You Ai

AU - Pepperl, David J.

AU - Regan, John W

AU - Stevens, Jeffrey C.

AU - Halpert, James R.

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N2 - 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 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-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 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-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 (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-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.

AB - 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 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-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 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-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 (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-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.

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