INVOLVEMENT OF CYTOCHROME-P-450 IN THE 15-ALPHA-HYDROXYLATION OF 13-ETHYL-GON-4-ENE-3,17-DIONE BY PENICILLIUM-RAISTRICKII

The 15 alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was i nvestigated. Cytochrome P-450 was shown to be involved in this reactio n. The steroid transformation was inhibited by carbon monoxide, metyra pone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15 alpha-hydroxylase was observed to be dependent on nicotinamide- adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the a ctivity was enhanced by a NADPH-regenerating system, indicating the in volvement of the NADPH-cytochrome c (P-450) reductase. This was furthe r confirmed by the inhibition of the hydroxylase activity in the prese nce of cytochrome c. No effect was observed in the presence of azide a nd antimycin A. Solubilized microsomes gave an absorption maximum at 4 53 nm in carbon monoxide difference spectrum, and showed a Type-I GD-b inding spectrum typically for cytochrome P-450 interaction with substr ate. First results about the inducibility of the enzymes involved in t he 15 alpha-hydroxylation of GD are shown. (C) 1997 Elsevier Science L td.