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J. Biochem, 1988, Vol. 103, No. 5 853-857
© 1988 Japanese Biochemical Society

research-article

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 Microsomes1

Shin-ichi Hayashi*, Tsuneo Omura**, Takeshi Watanabe*** and Kyuichiro Okuda*

*Department of Biochemistry, School of Dentistry, Hiroshima University Minami-ku, Hiroshima, Hiroshima 734
**Department of Molecular Biology, Graduate School of Medical Science Higashi-ku, Fukuoka, Fukuoka 812
***Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University Higashi-ku, Fukuoka, Fukuoka 812

Polyclonal antibody elicited in a rabbit against purified cytochrome P-450cc25, which catalyzes 25-hydroxylation of vitamin D3, inhibited not only 25-hydroxylation of cholecal-ciferol 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.

1This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


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