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The activity of purified bovine adrenocortical P-450(11)beta on the C18-steroid, 4-estrene-3,17-dione (19-norandrostenedione), is described. The major steroid products were separated by HPLC and identified by GC-MS, and 1H- and 13C-NMR as 11 beta-, 18- and 6 beta-hydroxylated derivatives of 19-norandrostenedione. The turnover numbers of the 11 beta-, 18- and 6 beta-hydroxylase reactions were 45, 7.5 and 1.9 (mol/min/mol of P-450(11)beta), respectively, with a common Km of 44 microM. All of these activities required the presence of the electron donating system consisting of NADPH, adrenal ferredoxin (adrenodoxin) and its reductase. These findings provide additional insights into the versatile catalytic roles of P-450(11)beta in the adrenal cortex, in which it may act on C18-19-nor-steroids in addition to its known activities on C21- and C19-steroids.

The substrate specificity of 11 beta-hydroxylase of Cochliobolus lunatus was studied and a close parallelism to the results obtained with 11 alpha-hydroxylase of Rhizopus nigricans was observed. It was found that the cell wall does not differentiate between the steroid substrates used and the absence of the cell wall increases the bioconversion.

Steroids with nonpolar side chains with 2, 4 and 8 C atoms were used as substrates for the 11 alpha-hydroxylase of Rhizopus nigricans. Their bioconversion was compared to that of progesterone, which was found to be far the best substrate giving the highest total bioconversion. 3-keto-4-ene steroids with nonpolar side chains were converted to their hydroxylated products in a small yield or not at all. The absence of an oxygen function in the side chain did not affect the regio-specificity of the hydroxylation, but resulted in a much lower total bioconversion. The strong effect of the oxygen function and of the length of the side chain on hydroxylation with the 11 alpha-hydroxylase of Rhizopus nigricans was demonstrated.

The effect of thyroidectomy on corticosterone and aldosterone biosynthesis, in rat adrenal cortex, were investigated in vitro. Thyroidectomy slowed down by about 30% the activity of microsomal 21 hydroxylase and mitochondrial 11 beta hydroxylase, the two stages leading to corticosterone formation from progesterone. The plasma corticosterone concentration also diminished in thyroidectomized rat. The mitochondrial enzyme system catalyzing the conversion of corticosterone into aldosterone included both 18 hydroxylase and 180H dehydrogenase. In the absence of thyroid hormone, values of both enzyme activity slowed down by about 30%. Thus thyroidectomy induced a general decrease in all mitochondrial hydroxylation processes requiring NNADPH as energetic cofactor. Mitochondrial NADPH was mainly synthesized by the activity of NADP+ malic enzyme and pyridine nucleotide transhydrogenase; the activity of both enzymes was determined in submitochondrial particles from adrenal cortex of normal and thyroidectomized rats. The activity of NADP+ malic enzyme and non energy-dependent transhydrogenase in the direction of NADPH formation dropped significantly after thyroidectomy, thus reducing the amount of NADPH available for hydroxylating mechanisms. The results should be compared to the large rise in th cytosolic malic enzyme level caused by thyroidectomy. The latter also ensured that the transhydrogenase reaction did not vary in the direction of NADP+ formation. Further, the drop in NADPH synthesis after thyroidectomy was coupled with a decrease in phosphorylation rate of added ADPH by adrenal cortex mitochondria of thyroidectomized rat. These findings might account for the general decline in the energy-dependent processes involved in steroidogenesis, a decline mainly due to the lowering of the H+ ions gradient driving ATP and NADPH synthesis through electron transfer.

19-Oxo-11-deoxycorticosterone was incubated with a cytochrome P-450(11)beta-reconstituted system, and the metabolites were analyzed by high performance liquid chromatography(HPLC). The main product found after chromatography was collected and treated with diazomethane. HPLC and 1H-NMR analysis of the methylated derivative indicated that it was 19-oic-11-deoxycorticosterone methyl ester. When 19-oic-11-deoxycorticosterone was stored at -20 degrees C for 1 month, it was spontaneously converted to other steroids. Structural analysis of the main degradation product indicated that it was 19-nor-11-deoxycorticosterone. These results suggest that the conversion of 19-oxo-11-deoxycorticosterone to 19-oic-11-deoxycorticosterone occurs through the P-450(11)beta-catalyzed reaction, and that the 19-oic-11-deoxycorticosterone thus formed is nonenzymatically converted into 19-nor-11-deoxycorticosterone.

Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose-dependent increase in the volume of zona fasciculata cells of dexamethasone-administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone-suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata.

Cholesterol side-chain cleavage activity (cholesterol SCC) in a mitochondrial preparation is increased by calcium in a sigmoidal manner. A 5-10-fold increase is obtained and an effect may be seen at 20 microM CaCl2. ADP inhibits the stimulation by calcium with a shift of the sigmoid curve to the right and 10 microM ADP results in a 4-fold increase in the amount of CaCl2 required to obtain one-half the maximal stimulation value. The inhibition is specific for ADP and inhibition by ATP is due to the formation of ADP. The characteristics of the calcium-ADP modulation are such that a suitable ADP-inhibited level of cholesterol sidechain cleavage activity will be stimulated by a given increment of calcium to a greater extent than in the absence of the added ADP. Steroid 11 beta-hydroxylation is also stimulated in a sigmoidal manner by calcium and this stimulation is inhibited by ADP. The 11 beta-hydroxylation, however, is less sensitive to calcium and ADP so that changes in cholesterol SCC are obtained at concentration of calcium and ADP where minimal effects on 11 beta-hydroxylation are found. Calmodulin-like activity is present in the mitochondrial preparation. No evidence, however, for a role for calmodulin in the calcium-ADP effects could be obtained, but the possibility of its involvement cannot be excluded. The calcium-ADP modulations are of a magnitude and take place at sufficiently low concentrations to suggest a physiological role in the regulation of mitochondrial steroidogenesis.

Zona glomerulosa and zonae fasciculata-reticularis of bovine adrenal cortex were separated by our improved method using a remodeled microtome. A considerable amount of fresh mitochondria and microsomes was quickly obtained. Specific content of cytochrome P-450 in the mitochondria was higher in zonae fasciculata-reticularis than in zona glomerulosa. When metyrapone was added to the mitochondria and the drug-induced difference spectra were measured, there appeared two phases in the titration curve; the first phase was saturated at a concentration of 3 microM and the second phase appeared at concentrations higher than 10 microM. The first phase seemed to be due to the substrate-free P-450(11) beta and the second phase to the cholesterol-bound P-450scc. From these spectral studies contents of cytochrome P-450scc and P-450(11) beta were estimated, and those were found to be equal for each zone. From similar studies on substrate-induced difference spectra, there seemed to be no difference between two zones in the affinity of cytochrome P-450(11) beta for deoxycorticosterone and 11-deoxycortisol. This was also the case with 11 beta-hydroxylase activity and substrate specificity for deoxycorticosterone and 11-deoxycortisol.

Three groups of 3-keto-4-ene steroids with different side chains were used as substrates for the induced 11 alpha-hydroxylase of Rhizopus nigricans. The highest total bioconversion as well as the highest yield of 11 alpha-hydroxylated product is found using progesterone as substrate. By changing the polarity of the side chain, much higher yields of 6 beta- and 7 beta-hydroxylated products relative to 11 alpha-hydroxylated product are obtained. Our results thus provide evidence for the importance of the side chain in steroid-enzyme interactions.

The effects of a long-term ACTH administration on the morphology of rat adrenocortical cells and the activity of some enzymes of steroid synthesis contained in them were examined by stereological and biochemical techniques. It was demonstrated that ACTH provokes time-dependent increases in the surface area per cell of smooth endoplasmic reticulum and mitochondrial cristae, which are tightly parallelled by enhancements in the activity per cell of delta 5-3 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxylase, respectively.

The effects of nicotine, cotinine and anabasine on rat adrenal steroidogenesis were examined by spectral and enzymatic techniques. The addition of nicotine, cotinine or anabasine to preparations of rat adrenal mitochondria produced type II cytochrome P-450 difference spectra. The addition of nicotine or anabasine, but not cotinine, to rat adrenal microsomes yielded type II cytochrome P-450 difference spectra. Nicotine and anabasine competitively inhibited rat adrenal mitochondrial 11 beta-hydroxylase and microsomal 21-hydroxylase. Cotinine competitively inhibited mitochondrial 11 beta-hydroxylase, but did not inhibit microsomal 21-hydroxylase. The apparent enzymatic inhibition constants for cotinine, nicotine, anabasine and metyrapone inhibition of the mitochondrial 11 beta-hydroxylase were 32, 96, 120 and 74 microM respectively. These studies suggest that components of cigarette smoke may alter patterns of adrenal steroidogenesis.