[Gene structure and gene expression regulation of cytochrome P-450 related to steroidogenesis].
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Amino Acid Sequence↗
Biomedical subjects
H Inano
Publications and source records attributed to H Inano.
Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co.
Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.
Adrenal Glands↗
Chemical modification of lysine residues at active-site of human placental estradiol 17 beta-dehydrogenase.
Estradiol 17 beta-dehydrogenase (EC 1.1.1.62.) activity was decreased by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent for modification of epsilon-amino moiety of lysine residues in a protein. The inactivation exhibited pseudo-first-order kinetics, and was protected by oxidyzed cofactors. Stoichiometric studies showed that the complete inactivation was caused by modification of one lysine residue per molecule of the enzyme. Differential modification with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), TNBS and dithiothreitol (DTT) indicated that the residues of lysine and cysteine were located at the active-site and played an essential role in the catalytic function of the estradiol 17 beta-dehydrogenase.
17-Hydroxysteroid Dehydrogenases↗
Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells.
The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.
3-Hydroxysteroid Dehydrogenases↗
Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).
The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.
Aldehyde-Lyases↗
Developmental changes of steroidogenic enzyme activities in the embryonic gonads of the chicken: the sexual difference.
Steroidogenic enzyme activities in the left ovary and the testes of 9- to 15-day-old chicken embryos were measured, and development of the activities was compared between sexes. Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary and in the testis was comparable, and did not change throughout the period examined. Activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the ovary was similar to or higher than that in the testis, depending on substrates employed. In both gonads, 17 beta-HSD activity did not change or tended to decrease from 9 to 15 days of development. On the other hand, activities of 17 alpha-hydroxylase, C-17--C-20 lyase in the ovary were three to eight times those in the testis, and aromatase activity in the ovary was definitely higher than that in the testis at all stages examined. The activities of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase significantly increased from 9 to 11 days only in the ovary. From 13 to 15 days, the activities of 17 alpha-hydroxylase and C-17--C-20 lyase markedly increased only in the testis. These results suggest that, in the gonads of developing chicken embryos, there are sexual differences in the regulation of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase activities.
17-Hydroxysteroid Dehydrogenases↗
Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate.
Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.
Animals↗
[Heart disease and bronchial circulation].
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Animals↗
Testicular and adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.
The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.
3-Hydroxysteroid Dehydrogenases↗
Conversion of androstenedione to 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-4-androsten-17-one by the testicular microsomal fraction of Sprague-Dawley rats.
When androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7 alpha-hydroxyandrostenedione. The metabolites were identified as 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one catalyzed by the 3 beta-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.
Acetylation↗
Ketoconazole as a possible universal inhibitor of cytochrome P-450 dependent enzymes: its mode of inhibition.
Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450 dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid 17 alpha-hydroxylase and C17-20 lyase in rat and human testes, 16 alpha-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of 10(-8)M for human testicular enzymes, while the order was 10(-7)-10(-6) M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450 and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450 dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.
Adenoma↗
Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and its relation to the ultrastructure of steroidogenic cells in immature and mature rat ovaries.
Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its relation to the ultrastructure of steroidogenic cells were examined in mature and immature rat ovaries. In mature (8-10 weeks old) rat ovaries, the theca interna cells of secondary as well as Graafian follicles, and the interstitial gland cells were all strongly stained with anti-17 beta-HSD antibody. However, granulosa cells, corpus luteum cells, oocytes and peritoneal epithelial cells were negative against this staining. In the ovaries of 1-week-old rats, all these cells were negative to immunostaining for 17 beta-HSD. In the ovaries of 2-week-old rats, the theca interna cells of secondary follicles and the interstitial gland cells showed a positive reaction for the 17 beta-HSD activity. Electron microscopic examination demonstrated the presence of characteristic structures for steroid secretory cells such as many lipid droplets, well developed smooth endoplasmic reticulum, and oval mitochondria with tubular cristae in the theca interna cell of secondary as well as Graafian follicles and in the interstitial gland cell of mature rat ovaries. In the ovaries of 1-week-old rats, all the theca cells of the primary and secondary follicles were fibroblast-like in their shape and fine structure, and typical interstitial cells were not recognized. In the 2-week-old rats, some of the theca interna cells and interstitial cells were well differentiated in ultrastructure, showing characteristic features for steroid secretory cells. These findings indicate that by 2 weeks after birth, theca interna cells and interstitial gland cells acquire the ability for testosterone production as seen in mature rat ovaries.
17-Hydroxysteroid Dehydrogenases↗
[Miliary tuberculosis complicated with disseminated intravascular coagulation].
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Acute Disease↗
[Gene and molecular structure of cytochrome p-450].
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Amino Acid Sequence↗
Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor.
Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.
Animals↗
Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane.
NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.
Animals↗
Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.
Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.
3-Hydroxysteroid Dehydrogenases↗
Reduced activity of androgen biosynthesis in the testes of rats with analbuminemia.
Steroid metabolism in Nagase Analbuminemia Rats (NAR), a mutant strain established from Sprague-Dawley rats, was studied. NAR are characterized by lack of serum albumin and hyperlipidemia. Total testosterone concentration in the serum of NAR was lower than that of normal rats, while the serum free testosterone, LH and FSH concentrations were similar. The half lives of 14C-labeled testosterone administered intravenously in NAR and normal Sprague-Dawley (SD) rats were 4.4 and 4.1 min, respectively. The plasma clearance rates of testosterone in NAR and normal rats were 34.7 and 39.1 ml/min per kg body weight. On Sephadex G-100 chromatography, a mixture of [3H]testosterone and normal rat serum gave two protein peaks eluted in the void volume and the albumin fraction, and the radioactivity was eluted all in the albumin fraction. In contrast, a mixture of [3H]testosterone and NAR serum gave a single protein peak eluted in the void volume and the radioactivity was mainly eluted with this protein peak. The association constants of testosterone to NAR and normal rat sera were 1.25 and 2.24 X 10(4) M-1. Enzyme activities related to the synthesis of testosterone by the testicular microsomal fractions of NAR and normal rats were examined. The activities of 3 beta-hydroxysteroid dehydrogenase, 5-ene-4-ene isomerase, 17 alpha-hydroxylase, C-17-C-20 lyase and 17 beta-hydroxysteroid dehydrogenase were lower in NAR than in normal rats. The activity for synthesis of testosterone from pregnenolone by the testicular microsomal fraction of NAR was about 40% of that of normal rats. These findings indicate that the low serum concentration of testosterone in NAR is mainly attributable to decreased biosynthesis of testosterone in the testes.
Androgens↗