Expression of Bovine 17α-Hydroxylase Cytochrome P-450 cDNA in Nonsteroidogenic (COS 1) Cells

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Abstract
Cortisol production requires the activity of only 17α-hydroxylase, whereas the formation of sex steroids requires both 17α-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17α-hydroxylase cytochrome P-450 (P-450 17α ), catalyzes both activities. By expression of bovine adrenocortical P-450 17α in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450 17α , it has now been established in situ that a single polypeptide chain does catalyze both the 17α-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17α-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17α-hydroxypregnenolone is the substrate than when 17α-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450 17α 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.

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