Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells.
Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells.
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DOI：
10.1126/science.3535074
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发表时间：
1986-12
期刊：
影响因子：
56.9
通讯作者：
M. X. Zuber;E. Simpson;M. Waterman
中科院分区：
文献类型：
--
作者：
M. X. Zuber;E. Simpson;M. Waterman
皮质醇的产生仅需要17 α-羟化酶的活性，而性类固醇的形成需要17 α-羟化酶和17，20-裂解酶的活性。对重组酶系统的研究表明，单一类固醇羟化酶，17 α-羟化酶细胞色素P-450（P-450（17）α），催化这两种活性。通过在COS 1（转化猴肾）细胞中表达牛肾上腺皮质P-450（17 α）（通常不含可检测到的P-450（17）α），现已原位证实，一条多肽链确实催化17 α-羟化酶和17，20-裂解酶反应。这种异源系统支持17 α-羟化的双羟烯醇酮和孕酮具有相同的效率，但当17 α-羟基双羟烯醇酮是底物时催化的17，20-裂解酶活性是当17 α-羟基孕酮是底物时催化的17，20-裂解酶活性的约5倍。为了在COS 1细胞中观察到这些活性，新合成的脱辅基细胞色素P-450（17）α必须结合血红素并插入内质网，以便内源性细胞色素P-450还原酶能够支持羟基化。因此，COS 1细胞是一个有用的系统，表达和研究各种形式的细胞色素P-450。
Cortisol production requires the activity of only 17 alpha-hydroxylase, whereas the formation of sex steroids requires both 17 alpha-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha), catalyzes both activities. By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450(17) alpha, it has now been established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17 alpha-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17 alpha-hydroxypregnenolone is the substrate than when 17 alpha-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450(17) alpha must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.