FUNCTIONAL AND EXPRESSION ANALYSIS OF OVINE STEROID 11-BETA-HYDROXYLASE (CYTOCHROME P450(11-BETA))

In this study, the ovine steroid 11 beta-hydroxylase (P450(11 beta) or CYP11B) cDNA previously reported by us (1) was transfected into COS-7 cells. Using H-3-11-deoxycorticosterone (H-3-DOC) as the substrate, a nd paper partition chromatography for separation of steroid products, the expressed enzyme was shown to catalyse the conversion of DOC to co rticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hy droxy-corticosterone (18-OH-B), and aldosterone (ALDO). These results suggest that the expressed ovine cDNA exhibited 11 beta-hydroxylase, 1 8-hydroxylase and aldosterone synthesis activities. The enzymatic acti vity of the enzyme was further analysed by adding unlabelled steroids to compete with H-3-DOC. The conversion of H-3-DOC to H-3-ALDO was inh ibited by the addition of excess DOC, B and 18-OH-DOC, indicating that all these steroids were potential substrates of the enzyme. The resul ts also demonstrated that 18-hydroxylation could occur before 11 beta- hydroxylation with this enzyme. However, the addition of excess cold 1 8-OH-B had no significant effect on the level of H-3-ALDO that was syn thesised. This result could imply that 18-OH-B is not an intermediate involved in the conversion of DOC to aldosterone, or, more likely, the enzyme substrate site is not accessible readily. Our results also ind icated that DOC was preferred to 18-OH-DOC as a substrate for the enzy me. We have demonstrated by hybridisation histochemistry using specifi c oligonucleotide probes that the corresponding P450(11 beta) RNA tran script was present in all zones in the sheep adrenal cortex. In summar y, we have shown that the enzyme encoded by the predominant P450(11 be ta) cDNA isolated from the sheep adrenocortical cDNA library has all t he enzymatic activities to biosynthesise ALDO from DOC. The correspond ing transcript of this ovine P450(11 beta) in cDNA was located through out the adrenal cortex and thus the inability of the zonae fasciculata -reticularis to secrete ALDO remains to be understood.