Kazutoshi Yanagibashi

Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.

Ascorbate as a source of reducing equivalents for the synthesis of aldosterone

The three steps in the synthesis of aldosterone (11 beta/18-hydroxylations and aldehyde synthetase) were examined in mitochondria from bovine glomerulosa and fasciculata to study the regulation of aldehyde synthetase. Ascorbate plus NADH shows synergism with malate in stimulating aldehyde synthetase without affecting 11 beta/-18-hydroxylations. The concentration of semidehydroascorbate reductase in mitochondria from glomerulosa is more than twice that from fasciculata. We propose that in glomerulosa, ascorbate provides a source of reducing equivalents that specifically support the last step in the synthesis of aldosterone.

Synthesis of Aldosterone by Mitochondria and Homogeneous 11β-Hydroxylase from Beef and Pig

It was found that homogeneous 11 beta-hydroxylase from bovine and porcine adrenals catalyzes the conversion of DOC to aldosterone. Mitochondria from both glomerulosa and fasciculata also convert DOC to aldosterone but glomerulosa is much more active than fasciculata. Cholate extracts of mitochondria from the two zones were equally active in converting DOC to aldosterone. Moreover all the enzyme activities of 11 beta-hydroxylase (including 18-hydroxylation and aldehyde synthetase) were precipitated by a polyclonal antibody raised in rabbit against the pure 11 beta-hydroxylase. It is concluded that in beef and pig a single adrenocortical 11 beta-hydroxylase is responsible for the synthesis of aldosterone. To determine the influence of the mitochondrial membrane from glomerulosa and fasciculata on the activities of 11 beta-hydroxylase we examined the activities of rotenone-insensitive reductase enzymes in mitochondria from the two zones. Semidehydroxyascorbate reductase and NADH-cytochrome C reductase activities are considerably more active in glomerulosa than in fasciculata mitochondria. Moreover ascorbate plus NADH (but not ascorbate alone) greatly increases the ability of malate and NADPH to support synthesis of aldosterone without affecting 11 beta- or 18-hydroxylations in mitochondria. It is proposed that maximal synthesis of aldosterone by adrenocortical mitochondria requires in addition to the usual electron transport system (NADPH- greater than ADR- greater than ADX- 11 beta-OHase) an auxilliary system in the outer mitochondrial membrane: NADH- greater than Fp- greater than cyt b- greater than semidehydroascorbate reductase.