Morohashi K, Nonaka Y, Kirita S, Hatano O, Takakusu A, Okamoto M, Omura T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
J Biochem. 1990 Apr;107(4):635-40. doi: 10.1093/oxfordjournals.jbchem.a123099.
Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.
利用两个P-450(11β)的cDNA克隆，即pcP-450-(11β)-2和pcP-450(11β)-3（森桥等人（1987年）《生物化学杂志》102卷，559 - 568页；桐田等人（1988年）《生物化学杂志》104卷，683 - 686页）构建表达质粒，并通过电穿孔法将其导入COS - 7细胞。通过免疫印迹、免疫荧光显微镜检查和免疫电子显微镜检查证实了P-450(11β)蛋白的表达及其在线粒体中的定位。以脱氧皮质酮（DOC）、脱氧皮质醇和皮质酮作为底物，测定所表达的P-450(11β)的酶活性。尽管两种P-450(11β)对DOC进行11 -、18 -和19 -羟化的活性几乎相等，但P-450(11β)-3从皮质酮生成18 -羟皮质酮和醛固酮的量大于P-450(11β)-2。
