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Original Article

Functional and expression analysis of ovine steroid 11ß-hydroxylase (Cytochrome P45011ß)

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Pages 325-347 | Published online: 07 Aug 2009
 

Abstract

In this study, the ovine steroid 11ß-hydroxylase (P45011ß or CYP11B) cDNA previously reported by us (1) was transfected into COS-7 cells. Using 3H-11-deoxycorticosterone (3H-DOC) as the substrate, and paper partition chromatography for separation of steroid products, the expressed enzyme was shown to catalyse the conversion of DOC to corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B), and aldosterone (ALDO). These results suggest that the expressed ovine cDNA exhibited 11ß-hydroxylase, 18-hydroxylase and aldosterone synthesis activities. The enzymatic activity of the enzyme was further analysed by adding unlabelled steroids to compete with 3H-DOC. The conversion of 3H-DOC to 3H-ALDO was inhibited by the addition of excess DOC, B and 18-OH-DOC, indicating that all these steroids were potential substrates of the enzyme. The results also demonstrated that 18-hydroxylation could occur before 11ß-hydroxylation with this enzyme. However, the addition of excess cold 18-OH-B had no significant effect on the level of 3H-ALDO that was synthesised. 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 indicated that DOC was preferred to 18-OH-DOC as a substrate for the enzyme. We have demonstrated by hybridisation histochemistry using specific oligonucleotide probes that the corresponding P45011ß RNA transcript was present in all zones in the sheep adrenal cortex.

In summary, we have shown that the enzyme encoded by the predominant P45011ß cDNA isolated from the sheep adrenocortical cDNA library has all the enzymatic activities to biosynthesise ALDO from DOC. The corresponding transcript of this ovine P45011ß cDNA was located throughout the adrenal cortex and thus the inability of the zonae fasciculata-reticularis to secrete ALDO remains to be understood.

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