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J. Biochem, 1988, Vol. 103, No. 5 858-862
© 1988 Japanese Biochemical Society

research-article

Expression of a Rat Liver Microsomal Cytochrome P-450 Catalyzing Testosterone 16{alpha}-Hydroxylation in Saccharomyces cerevisiae: Vitamin D3 25-Hydroxylase and Testosterone 16{alpha}-Hydroxylase Are Distinct Forms of Cytochrome P-450

Shin-ichi Hayashi*, Ken-ichirou Morohashi**, Hidefumi Yoshioka***, Kyuichiro Okuda* and Tsuneo Omura**

* Department of Biochemistry, School of Dentistry, Hiroshima University Minami-ku, Hiroshima, Hiroshima 734
** Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Higashi-ku, Fukuoka, Fukuoka 812
*** Institute of Comprehensive Medical Science, Fujita Gakuen Health University School of Medicine Toyoake, Aichi 470-11

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 × 105 molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16{alpha}- and 2{alpha}-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or l{alpha}-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5ß-cholestane-3{alpha}, 7{alpha}, 12{alpha}-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2{alpha}- and 16{alpha}-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16{alpha}-and 2{alpha}-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.


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