|Monoclonal antibodies of differentiating specificities as probes of cytochrome P450h (2C11).|
|PMID: 7681658 Owner: NLM Status: MEDLINE|
|A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2 alpha- and 16 alpha-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine N-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetamine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.|
|D E Ryan; P E Thomas; W Levin; S L Maines; S Bandiera; L M Reik|
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|Type: Journal Article|
|Title: Archives of biochemistry and biophysics Volume: 301 ISSN: 0003-9861 ISO Abbreviation: Arch. Biochem. Biophys. Publication Date: 1993 Mar|
|Created Date: 1993-04-23 Completed Date: 1993-04-23 Revised Date: 2005-11-17|
Medline Journal Info:
|Nlm Unique ID: 0372430 Medline TA: Arch Biochem Biophys Country: UNITED STATES|
|Languages: eng Pagination: 282-93 Citation Subset: IM|
|Department of Protein Biochemistry, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.|
|APA/MLA Format Download EndNote Download BibTex|
Antibodies, Monoclonal* / isolation & purification
Aryl Hydrocarbon Hydroxylases*
Benzphetamine / metabolism
Cytochrome P-450 Enzyme System / immunology*
Enzyme-Linked Immunosorbent Assay
Immunoglobulin A / immunology
Immunoglobulin G / immunology
Isoenzymes / immunology*
Mice, Inbred Strains
Microsomes, Liver / enzymology*, immunology
Rats, Inbred Strains
Steroid Hydroxylases / immunology*
|0/Antibodies, Monoclonal; 0/Epitopes; 0/Immunoglobulin A; 0/Immunoglobulin G; 0/Isoenzymes; 0/Molecular Probes; 156-08-1/Benzphetamine; 9035-51-2/Cytochrome P-450 Enzyme System; EC 1.14.-/Steroid Hydroxylases; EC 18.104.22.168/Aryl Hydrocarbon Hydroxylases; EC 22.214.171.124/CYP2C11 protein, rat; EC 126.96.36.199/Steroid 16-alpha-Hydroxylase|
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