Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation

James Halpert, John Yan Jaw, Celia Balfour, Eugene A Mash, Eric F. Johnson

Research output: Contribution to journalArticle

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Abstract

The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16α-hydroxylase activity, and three phenotypes of 6β-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1,21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6β-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B J strain. In contrast, in the microsomes from the B J rabbits, some inactivation of the 16α-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregnenolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.

Original languageEnglish (US)
Pages (from-to)462-471
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume264
Issue number2
DOIs
StatePublished - 1988

Fingerprint

Chlorinated Steroids
Hydroxylation
Mixed Function Oxygenases
Steroid 21-Hydroxylase
Liver
Cytochrome P-450 Enzyme System
Progesterone
Microsomes
Rabbits
Rifampin
Bearings (structural)
Phenotype
Enzyme Inhibitors
Substrates
NADP
Liver Microsomes
21,21-dichloropregnenolone

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation. / Halpert, James; Jaw, John Yan; Balfour, Celia; Mash, Eugene A; Johnson, Eric F.

In: Archives of Biochemistry and Biophysics, Vol. 264, No. 2, 1988, p. 462-471.

Research output: Contribution to journalArticle

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title = "Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation",
abstract = "The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16α-hydroxylase activity, and three phenotypes of 6β-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1,21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6β-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B J strain. In contrast, in the microsomes from the B J rabbits, some inactivation of the 16α-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregnenolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.",
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AB - The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16α-hydroxylase activity, and three phenotypes of 6β-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1,21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6β-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B J strain. In contrast, in the microsomes from the B J rabbits, some inactivation of the 16α-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregnenolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.

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