Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11β-hydroxylase) from bovine adrenal cortex

Abstract

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11β-hydroxylation (11β)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11β was able to incorporate phosphate from ATP in the presence of C ( K m = 7.5 μM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11β (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11β-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11β phosphorylation strikingly increases the P-450 11β-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11β-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.