Hilton A. Salhanick

The effect of steroid hydroxylase inhibitors on the rate of reduction of adrenal mitochondrial cytochrome P-450

Abstract When added to adrenocortical mitochondria, metyrapone, amphenone and aminoglutethimide induce type II difference spectra, and block both the initial rate of malate-linked cytochrome P-450 reduction and also the DOC-induced increase in this rate. Removing the -NH 2 group from aminoglutethimide shifts the spectrum to shorter wavelengths and reduces its effectiveness in blocking P-450 reduction. These compounds appear to block steroid hydroxylation by binding to oxidized P-450 and only very slowly allowing the P-450 to be reduced.

Modification of enzymatic activity and difference spectra of cytochrome P-450 from various sources by cholesterol side chain cleavage inhibitors

Several groups of compounds were tested for their ability to inhibit cholesterol side chain cleavage and induce spectral change in cytochrome P-450 from bovine corpus luteum, bovine adrenal cortex, and human placental mitochondria. The substances tested include: steroids, pyridines, glutarimides, anilines and imidazoles. Good correlation was found between spectral change and enzymatic inhibition, especially in the corpus luteum which has only a single P-450-linked steroid hydroxylase. The cholesterol side chain cleavage enzyme systems from each of the three sources appear to have similar affinities for the inhibitors, which adds further support to the concept that these cytochrome P-450s are functionally identical.

Differential effect of aminopterin on the action of estradiol and progesterone in monkeys

Abstract The influence of Aminopterin (4-amino pteroylglutamic acid) on the action of estradiol and progesterone on the reproductive tract was tested in twelve sexually mature Rhesus monkeys. The animals were castrated and given 10 μg estradiol daily for twenty days after which each received Aminopterin daily beginning with doses of 100 μg and increased after each succeeding ten days to 200 μg, 500 μg, and 1 mg., depending upon the symptoms of vitamin deficiency. During the Aminopterin treatment, six animals were given 10 μg estradiol daily, three received 10 μg estradiol and 1.5 or 2 mg. progesterone, and three 1.5 or 2 mg. progesterone. Three of the six animals on estrogen menstruated during treatment while bleeding did not occur in those receiving progesterone. The endometria of animals given progesterone showed full progestational development. Aminopterin inhibited the action of estrogen on the vagina, had no detectable influence on the response of the cervix to estrogen, and did not modify the response of these structures to progesterone. Therefore, under these conditions, Aminopterin showed a differential action on the effects of estrogen and progesterone on the female reproductive tracts of monkeys.

Amino acid composition of cytochrome P-450scc from bovine corpus luteum.

Summary The amino acid composition of cytochrome P-450 SCC , purified from bovine corpus luteal mitochondria was determined and expressed on the basis of the molecular weight 48,000, determined by SDS gel electrophoresis. The composition was similar to that reported for P-450 SCC from adrenocortical mitochondria, but different from P-450 11β from bovine adrenal mitochondria, P-450 LM from liver microsomes and P-450 cam from Pseudomonas putida . The molecule is composed of about 400 amino acids, 49% of which are non-polar.

Effects of cholesterol side chain cleavage inhibitors on 11β-hydroxylation: linkage of mitochondrial oxygenase systems

Potent inhibitors of cholesterol side chain cleavage were tested for inhibition of 11 beta-hydroxylation of 11-deoxycortisol by bovine adrenal cortex mitochondria. Compounds which inhibited 11 beta-hydroxylation were metyrapone, 4-phenylimidazole, 1-benzylimidazole, 17 beta-ureido - 1, 4- androstadien-3-one. SU-8000, 4-methylaminoglutethimide, and 20 alpha-hydroxycholestrol. Compounds which did not inhibit 11 beta-hydroxylation at concentrations of 0.5 mM were d-aminoglutethimide tartrate, 1-aminoglutethimide tartrate, N-methylaminoglutethimide, 16 alpha-methylpregnenolone, 16 beta-methylpregnenolone, 20-tolylpregnenediol, 16 alpha-chloropregnenolone-3-acetate, 16 alpha-benzyloxpregnenolone-3-acetate and cyanoketone. The results obtained indicate that aminoglutethimide and its congeners, the 16-halogenated and 16-benzoylated derivatives of pregnenolone and cyanoketone are specific inhibitors of cholesterol side chain cleavage enzyme. The two mitochondrial steroid oxyganase systems are linked through their competition for a single electron source.

Implication of Ligand Modified Spectra of Cytochrome P-450 Associated with Pregnenolone Synthesis in Mitochondria from Corpus Luteum

The implications of ligand modified spectra of cytochrome P-450 in mitochondria from corpus luteum are considered. Mitochondria from bovine corpus luteum contain a single cytochrome P-450 which oxidizes cholesterol to pregnenolone and isocaproaldehyde. These mitochondria and the cytochrome P-450 purified from these mitochondria yield Type I spectral changes with substrates, reverse Type I spectral changes with certain steroid substrates, steroid products and unrelated steroid ligands. Nitrogenous ligands yield Type II spectral changes. Mitochondrial and purified cytochrome P-450 preparations are equivalent in this study. The inhibitory effects on the cholesterol monooxygenase are directly related to the spectral changes induced by Type II ligands. Lastly, it is suggested that a similar relationship exists with reverse Type I ligands.

Relaxation of Pubic Symphysis of the Mouse during the Estrous Cycle and Pseudopregnancy.

Summary X-ray photographs were taken of the pubic symphyses of mice at various stages of the estrous cycle and the separations of the pubic bones were measured. Significant relaxation occurred during estrus. No change in separation could be detected during artificially induced pseudopregnancy even when the uteri were stimulated to produce deciduomata.