Biomaterial Database

Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450: P-450 form specificities and use as probes for cytochrome P-450-dependent steroid hydroxylations. 1987

D J Waxman, and D P Lapenson, and S S Park, and C Attisano, and H V Gelboin

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008297	Male		Males
D008862	Microsomes, Liver	Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough.	Liver Microsomes,Liver Microsome,Microsome, Liver
D010105	Oxygenases	Oxidases that specifically introduce DIOXYGEN-derived oxygen atoms into a variety of organic molecules.	Oxygenase
D003577	Cytochrome P-450 Enzyme System	A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism.	Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D005779	Immunodiffusion	Technique involving the diffusion of antigen or antibody through a semisolid medium, usually agar or agarose gel, with the result being a precipitin reaction.	Gel Diffusion Tests,Diffusion Test, Gel,Diffusion Tests, Gel,Gel Diffusion Test,Immunodiffusions,Test, Gel Diffusion,Tests, Gel Diffusion
D000072467	Cytochrome P450 Family 2	A cytochrome P450 enzyme family that includes members which function in the metabolism of STEROIDS; COUMARINS; and NICOTINE.	CYP2 Enzymes,CYP2 Family
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D000911	Antibodies, Monoclonal	Antibodies produced by a single clone of cells.	Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D001189	Aryl Hydrocarbon Hydroxylases	A large group of cytochrome P-450 (heme-thiolate) monooxygenases that complex with NAD(P)H-FLAVIN OXIDOREDUCTASE in numerous mixed-function oxidations of aromatic compounds. They catalyze hydroxylation of a broad spectrum of substrates and are important in the metabolism of steroids, drugs, and toxins such as PHENOBARBITAL, carcinogens, and insecticides.	Microsomal Monooxygenases,Xenobiotic Monooxygenases,Hydroxylases, Aryl Hydrocarbon,Monooxygenases, Microsomal,Monooxygenases, Xenobiotic