Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine

Immunochemical and Radiochemical Analyses of the Protective Effects of Glutathione

Uma Sharma, Elizabeth S. Roberts, Ute M. Kent, S. Michael Owens and Paul F. Hollenberg
Drug Metabolism and Disposition February 1997, 25 (2) 243-250;
Uma Sharma
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elizabeth S. Roberts
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ute M. Kent
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
S. Michael Owens
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul F. Hollenberg
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Immunochemical and Radiochemical Analyses of the Protective Effects of Glutathione

Abstract

Phencyclidine (PCP) inactivates the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of P4502B1 in a reconstituted system containing NADPH-cytochrome P450 (P450) reductase (reductase) andl-α-phosphatidylcholine, dilauroyl in a time-, concentration-, and NADPH-dependent manner. Catalytic activity of the enzyme could not be restored upon reconstitution with fresh reductase, indicating that the effect was on the P450 and not on the reductase. Although the kinetics suggested that PCP would be classified as a classical mechanism-based inactivator, protection against inactivation of P450 by PCP by the presence of an exogenous nucleophile, such as glutathione (GSH), indicated otherwise. There was no loss of spectrally detectable P450 associated with inactivation either in the presence or absence of GSH. When radiolabeled PCP was used to inactivate the enzyme and the reaction mixture analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found to be associated with P450, reductase, and catalase that had been added to protect against oxidative damage. When GSH was included in the reaction mixtures, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked decrease in the binding to all three proteins. Correspondingly, analysis of the components of the inactivated sample by reversed-phase HPLC demonstrated that radioactivity was associated with P450, reductase, and catalase, and that there was a marked decrease in the labeling of all three proteins in the presence of GSH. The stoichiometry of binding of radiolabeled PCP to the proteins in the incubation mixture in the absence of GSH was 4:1. In the presence of GSH, no significant amount of radioactivity was incorporated into the proteins. An anti-PCP metabolite antibody was used to detect PCP metabolite adducts bound to the inactivated enzyme by Western blot analysis. The antibody recognized adducts bound to P450, reductase, and catalase. In the presence of GSH, there was a decrease in immunoreactivity, although binding of PCP to all three proteins was still detected. Because the added nucleophile protects against inactivation and protein labeling by PCP, these data suggest that the reactive intermediate may escape from the active site and attack other sites on the P450, as well as other proteins in the milieu.

Footnotes

  • Send reprint requests to: Dr. Paul F. Hollenberg, Department of Pharmacology, 2301 Medical Sciences Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632.

  • This study was supported in part by Grant CA16954 from the National Cancer Institute (U.S. Public Health Service) (to P.F.H.), by Grant DA04136 (National Institute on Drug Abuse), and by a Research Scientist Development Award K02 DA00110 (to S.M.O.).

  • Abbreviations used are::
    PCP
    phencyclidine
    P450
    cytochrome P450
    P4502B1
    the major P450 form from liver microsomes of phenobarbital-treated rats
    DLPC
    l-α-phosphatidylcholine, dilauroyl
    EFC
    7-ethoxy-4-trifluoromethylcoumarin
    HFC
    7-hydroxy-4-trifluoromethylcoumarin
    ECL
    enhanced chemiluminescence
    reductase
    NADPH cytochrome P450 oxidoreductase
    BSA
    bovine serum albumin
    KCN
    potassium cyanide
    GSH
    glutathione
    TFA
    trifluoroacetic acid
    SDS-PAGE
    sodium dodecyl sulfate-polyacrylamide gel electrophoresis
    PCHAP
    5-[N-(1′-phenylcyclohexyl)amino]pentanoic acid
    TTBS
    Tris–Tween-buffered saline
    • Received August 8, 1996.
    • Accepted November 19, 1996.
  • The American Society for Pharmacology and Experimental Therapeutics
View Full Text

 

DMD articles become freely available 12 months after publication, and remain freely available for 5 years. 

Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page. 

 

  • Click here for information on institutional subscriptions.
  • Click here for information on individual ASPET membership.

 

Log in using your username and password

Forgot your user name or password?

Purchase access

You may purchase access to this article. This will require you to create an account if you don't already have one.
PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition
Vol. 25, Issue 2
1 Feb 1997
  • Table of Contents
  • Index by author
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine

Uma Sharma, Elizabeth S. Roberts, Ute M. Kent, S. Michael Owens and Paul F. Hollenberg
Drug Metabolism and Disposition February 1, 1997, 25 (2) 243-250;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine

Uma Sharma, Elizabeth S. Roberts, Ute M. Kent, S. Michael Owens and Paul F. Hollenberg
Drug Metabolism and Disposition February 1, 1997, 25 (2) 243-250;
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Ontogeny of CPPGL
  • Expression of AKR and SDR Isoforms in the Human Intestine
  • Metabolism of Lufotrelvir in Humans
Show more Article

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2023 by the American Society for Pharmacology and Experimental Therapeutics