Monoclonal antibodies to rabbit liver cytochrome P-450LM2 and cytochrome P-450LM4.
Monoclonal antibodies were prepared from hybridoma clones isolated by the fusion of myeloma cells and spleen cells derived from mice immunized with either purified rabbit liver microsomal cytochrome P-450LM2 or cytochrome P-450LM4. Seven hybridoma clones produced three kinds of monoclonal antibodies to P-450LM2. The first class bound, precipitated, and inhibited the enzyme activity of P-450LM2 for both benzo[a]pyrene hydroxylation and 7-ethoxycoumarin deethylation. The other two classes either bound and precipitated or only bound the enzyme. These monoclonal antibodies to P-450LM2 showed a precipitin reaction and inhibition of enzyme activity that was specific for cytochrome P-450LM2. Thus, they did not react with or inhibit the enzyme activity of the other isozyme cytochrome P-450LM4, Fraction 1 or Fraction 7. All of the monoclonal antibodies formed against P-450LM2 were mouse immunoglobulin (Ig) subclass IgG1. The most effective monoclonal antibody strongly inhibited the formation of oxygenated metabolites of benzo[a]pyrene at various positions as well as the deethylation of 7-ethoxycoumarin. Four hybridomas were isolated which produced monoclonal antibodies to P-450LM4. One of the four was of the IgM class and three were of the IgG1 type. The four monoclonal antibodies bound to P-450LM4 but did not precipitate the enzyme, and did not bind to P-450LM2. The monoclonal antibody P-450LM4 complexes interacted with protein A, and the enzyme activity for benzo[a]pyrene hydroxylation could be removed by centrifugation. The high specificity and monoclonality of these antibodies suggest their potential usefulness for studying the genetics, regulation, and roles of the different isozymes of P-450LM in drug and carcinogen metabolism.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001201:Ascites / Q000201:enzymology; D001580:Benzopyrenes / Q000378:metabolism; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology* / Q000378:metabolism; D006825:Hybridomas / Q000201:enzymology; D005779:Immunodiffusion; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D010105:Oxygenases / Q000378:metabolism; D011817:Rabbits
D000911:Antibodies, Monoclonal; D001580:Benzopyrenes; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase