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Inactivation of Escherichia-coli expressed rabbit cytochrome-P-450 2C enzymes by 17-beta-substituted steroids

Academic Article
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Overview

authors

  • Klekotka, P. A.
  • Richardson, T. H.
  • Johnson, Eric
  • Halpert, J. R.

publication date

  • 1995

journal

  • Journal of Pharmacology and Experimental Therapeutics  Journal

abstract

  • The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

subject areas

  • Animals
  • Aryl Hydrocarbon Hydroxylases
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System
  • Escherichia coli
  • Kinetics
  • Mutagenesis
  • Pregnenolone
  • Progesterone
  • Rabbits
  • Steroid 16-alpha-Hydroxylase
  • Steroid Hydroxylases
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Identity

International Standard Serial Number (ISSN)

  • 0022-3565

PubMed ID

  • 7562521
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Additional Document Info

start page

  • 1450

end page

  • 1455

volume

  • 274

issue

  • 3

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