18‐Hydroxylation of Deoxycorticosterone by Reconstituted Systems from Rat and Bovine Adrenals

Abstract

18-Hydroxylation of deoxycorticosterone was studied with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P-450 reductase preparation from bovine adrenals. In some experiments, a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca²⁺ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-450 reductase was to some extent rate-limiting. The rate of 18–hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rate of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11β-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11β-and 18-hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11β-and 18-hydroxylation of deoxycorticosterone.