Studies on steroid hydroxylations in the adrenal cortex

Methods were developed for the preparation of large quantities of bovine adrenal cortex mitochondria. Adrenodoxin and adrenodoxin reductase, two components of the steroid hydroxylases found in these mitochondria, were isolated and examined. These two enzymes are responsible for the transfer of reducing equivalents from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to adrenal mito¬ chondrial cytochrome P450. Some of the factors which may affect their functioning in the supply of electrons for corticosteroid hydroxylations were also investigated. The amino acid composition of adrenodoxin reductase, a flavoprotein, was determined. Experiments showed that the protein had an isoelectric point near pH 8.9. The maximal rate of electron transfer to dichlorophenolindophenol (DCPIP) took place at pH 6.5. The coenzyme, flavinadenine dinucleotide (FAD), was found to dissociate from the protein in the course of enzyme purification. This resulted in the enzyme losing its activity which was however readily regained on addition of FAD. Addition of excess FAD however resulted in inhibition of the activity of adrenodoxin reductase in reducing DCPIP and of steroid hydroxylation by unfractionated mitochondrial enzyme extracts. It is suggested that this is due to the excess FAD being reduced and autoxidised and thus diverting the flow of electrons. The reduction of FAD by NADPH in the presence of adrenodoxin reductase was demonstrated under anaerobic conditions. Gel-filtration of adrenodoxin showed its molecular weight to be about 12,000. Electro focussing of this protein resulted in its denaturation at pH 5.0. Investigations as to the possible existence of different species of bovine adrenodoxin did not yield any decisive evidence. The amino acid composition of the enzyme closely resembled published figures except in the values for the glutamic and aspartic residues. • The reconstitution of the adrenal mitochondrial steroid hydroxylating enzyme system from purified components confirmed that high proportions of adrenodoxin relative to cytochrome P450 gave maximal in vitro hydroxylation rates. The rates of hydroxylation by these reconstituted systems were influenced by changes in ionic strength or the inclusion of bovine serum albumin in the assay medium. Excess NADPH or large excesses of adrenodoxin inhibit the reduction of cytochrome c by mitochondrial cytochrome P450 reductase (adrenodoxin plus adrenodoxin reductase). Excess concentrations of NADPH inhibited deoxycorticosterone (DOC) 11 ji -hydroxylation while the Km values for NADPH for DOC 11yj-hydroxylation and cholesterol side-chain cleavage to pregnenolone appear to be different. The adrenal cortical HAD-kinase is located in the cytoplasm of the cell. The specific activity of the enzyme is comparable to that of the enzyme in other tissues and was not found to be affected by treatment with ACTH (in vivo or in vitro) or 3**5' cyclic MP (in vitro). The principal effect of oxidised or reduced glutathione on DOC 11^3-hydroxylation by intact adrenocortical mitochondria was a marked inhibition. Evidence has been obtained that adrenodoxin and adrenocortical mitochondrial cytochrome P450 can interact with phospholipids to form isooctane-soluble proteolipids, A molecule of adrenodoxin appears to complex with four molecules of phospholipid. The phospholipid complex formed with cytochrome P450 contains 26 ug phosphorus each as phosphatidylethanolamine and as lecithin per mg protein extracted into the isooctane phase. The proteolipid formed by the haemprotein gives a spectral extinction maximum at 420 nm in isooctane solution following reduction with dithionite and treatment with carbon monoxide. The interaction of adrenodoxin and adrenodoxin reductase has been observed spectrophotometrically. This interaction is affected by inorganic salts such as sodium chloride as is the interaction of adrenodoxin and cytochrome P450. The ionic strength also affects the reduction of cytochrome c by adrenodoxin plus adrenodoxin reductase and DOC 11 jl -hydroxylation by the mitochondrial steroid hydroxylase. The different effects of various monovalent anions and cations on DOC 11y3-hydroxylation were found to be directly related to the ionic radius of each ion. Several dipolar ions known to affect the dielectric constant of aqueous solutions were tested and found to affect the rate of steroid hydroxylations by adrenocortical mitochondrial enzyme. It is possible that the unequal effects of various monovalent inorganic ions on DOC 11 y9-hydroxylation are related to the effects of their ionic radii on the known variation of the dielectric constant with distance from a monovalent ion. The hypothesis is advanced that adrenodoxin, an iron-sulphur protein component of mitochondrial but not microsomal steroid hydroxylases, is a regulatory particle. Its function of electron transfer for mitochondrial corticosteroid hydroxylations is effected as a shuttle between adrenodoxin reductase and cytochrome P450. The rate limiting nature of this step could be a reason for the increased steroid hydroxylation observed in in vitro enzyme assays on addition of purified adrenodoxin which is not linked by phospholipid bonds to a distinct functional enzyme unit. The shuttling movement or (xv) oscillation of adrenodoxin is thought to be affected by the composition of the intramitochondrial contents. This could constitute a regulatory mechanism of the biosynthesis of mineralocorticoids and other corticosteroids