Studies on the microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: isolation, characterization, and solubilization of the hydroxylase system

Abstract

A suitable procedure for the preparation of cell-free extract with high 11α-hydroxylase activity from the induced vegetative cell cultures of Aspergillus ochraceus was developed. The presence of EDTA (10 mM), glycerol (10%), and dithiothreitol (5 mM), in the grinding medium was found to be necessary for optimal hydroxylase activity. Although the pH optimum of the hydroxylase was found to be 7.7, the ideal pH for preparing the cell-free extract from mycelium was 8.3. Microsomes (2.0 mg) isolated from the cell-free extract hydroxylated progresterone in very high yields (85–90% in 30 min) in the presence of NADPH and O2. Cytosolic involvement in the hydroxylation reaction was not noticed. The apparent Km for NADPH and progesterone were 0.052 and 0.625 mM, respectively. The hydroxylase activity was inhibited by metyrapone, carbon monoxide, SKF-525A, and p-chloromercuribenzoate indicating the involvement of cytochrome P-450 system in the reaction. Inhibition of the hydroxylase activity by cytochrome c and the presence of significant levels of NADPH–cytochrome c reductase in the microsomal fraction suggested that the reductase could be one of the components of the hydroxylase system. The membrane-bound hydroxylase was solubilized using sodium cholate. Solubilized membrane fraction contained considerable levels of cytochrome P-450 and NADPH–cytochrome c reductase, besides hydroxylase activity.