Open Access18‐Hydroxylation of Deoxycorticosterone by Reconstituted Systems from Rat and Bovine Adrenals
Author(s) -
BJÖRKHEM Ingemar,
KARLMAR KarlEric
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb03915.x
Subject(s) - hydroxylation , chemistry , endocrinology , medicine , biology , biochemistry , enzyme
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 Ca 2+ 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.