Immunochemical evidence for the catalysis of vitamin D3 25-hydroxylation and testosterone 16 alpha-hydroxylation by homologous forms of cytochrome P-450 in rat liver microsomes.
Polyclonal antibody elicited in a rabbit against purified cytochrome P-450cc25, which catalyzes 25-hydroxylation of vitamin D3, inhibited not only 25-hydroxylation of cholecalciferol and 1 alpha-hydroxycholecalciferol, but also 16 alpha- and 2 alpha-hydroxylation of testosterone catalyzed by the purified P-450cc25 preparation. Antibody inhibition experiments with microsomes revealed that most 16 alpha- and 2 alpha-hydroxylation of testosterone and most 25-hydroxylation of cholecalciferol by male rat liver microsomes were catalyzed by P-450cc25. In order to examine the identity of cholecalciferol 25-hydroxylase and testosterone 16 alpha-hydroxylase, monoclonal antibodies recognizing three different epitopes of P-450cc25 were prepared from hybridoma clones produced by fusion of mouse myeloma cells (P3X63Ag8U1) with the spleen cells of immunized BALB/c mouse. All of these monoclonal antibodies inhibited both 25-hydroxylation of 1 alpha-hydroxycholecalciferol and 16 alpha-hydroxylation of testosterone by purified P-450cc25. These observations suggested that immunochemically indistinguishable form(s) of cytochrome P-450 catalyzed both reactions.
Hayashi
S
S
Department of Biochemistry, School of Dentistry, Hiroshima University.
Omura
T
T
Watanabe
T
T
Okuda
K
K
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002384:Catalysis; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006900:Hydroxylation; D007120:Immunochemistry; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; C450239:CYP2C3 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster; D053493:Cholestanetriol 26-Monooxygenase; C506832:Cyp27a1 protein, mouse