Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.     

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.     

Mild adrenal 3 beta-hydroxysteroid dehydrogenase deficiency with hyperaldosteronism

The patient was admitted to our hospital at 19 and again at 22-yr of age for hirsutism and hypertension. Her baseline and ACTH-stimulated plasma 17-hydroxy pregnenolone, dehydroepiandrosterone and dehydroepiandrosterone sulfate were increased whereas plasma 17-hydroxy progesterone and androstenedione were normal and responded poorly to ACTH. Plasma deoxycorticosterone, corticosterone and cortisol baseline levels were normal, and they responded normally to ACTH. The plasma aldosterone concentration (PAC) was always high and responded well to ACTH, angiotensin III and furosemide-upright stimulation. However, plasma renin activity (PRA) was normal or slightly high, and responded normally to furosemide-upright stimulation and fluorohydrocortisone suppression. Dexamethasone (2 mg/day) for 1-2 weeks suppressed the androgens, cortisol and corticosterone levels. PRA and PAC were suppressed temporally, but PRA returned to normal and PAC to be a high level after 2 weeks of dexamethasone administration. Blood pressure was also reduced temporally but returned to a high level after 2 weeks of dexamethasone. These results indicate that primary aldosteronism and dexamethasone-suppressible hyperaldosteronism were not likely to be present, and unknown aldosterone stimulating factors which potentiated the action of endogenous angiotensin II or ACTH might be responsible for the hyperaldosteronism in this patient. We conclude that this patient had a mild and non-salt losing 3 beta-HSD deficiency in the zona reticularis with normal fasciculata and high glomerulosa function.     

Evidence for distinct dehydrogenase and isomerase sites within a single 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein.

Complementary DNA encoding human 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3 beta-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3 beta-HSD protein. In addition, using 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta, 17 beta-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Effect of a peroxisome proliferator on 3 beta-hydroxysteroid dehydrogenase

To better understand the changes that occur following exposure to peroxisome proliferators, we utilized mRNA differential display and microarray to screen for peroxisome proliferator target genes apart from those involved in lipid metabolism in male C57B6 mice by using the ubiquitous plasticizer, di(2-ethylhexyl)phthalate (DEHP). One noted change was the dose-dependent suppression of the mouse hormone metabolizing 3 beta-hydroxysteroid dehydrogenase V (HSD3b5), which is specifically expressed in the male mouse liver. Northern analysis showed that HSD3b5 mRNA levels decreased dramatically upon one-day exposure to 2.0% dietary DEHP, and were nearly undetectable by one week of treatment. Food restriction also significantly suppressed HSD3b5 expression; however, in this case the suppression was delayed and to a lesser extent. Another mouse 3 beta-hydroxysteroid dehydrogenase, HSD3b4, predominantly expressed in kidneys, was also regulated by DEHP and food restriction. The sex-specific gene, HSD3b5, was affected more by DEHP and food restriction than the tissue-specific gene, HSD3b4.     

Histochemical study on adrenal delta 5-3 beta-hydroxysteroid dehydrogenase activity in cadmium treated toad (Bufo melanostictus).

Effect of a single subcutaneous injection of cadmium chloride at the dose of 0.5 mg/toad on adrenal delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) was observed after 7 days. The activity of delta 5-3 beta-HSD was measured histochemically. The experiments indicate that cadmium chloride resulted in a significant decrease in the activity of adrenal delta 5-3 beta-HSD in toad during breeding season (June-July).     

Effect of ketoconazole on placental aromatase, 3 beta-hydroxysteroid dehydrogenase-isomerase and 17 beta-hydroxysteroid dehydrogenase

Ketoconazole, an orally-active, broad spectrum mycotic agent, was shown to inhibit in vitro human placental microsomal aromatase but was without effect on 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities. The Km of placental aromatase for testosterone was 30 +/- 1.1 nmol/l (mean +/- SEM, n = 6). Inhibition (determined by Lineweaver-Burk plot) was non-competitive with respect to substrate with a Ki value of 3.0 +/- 1.4 mumol/l (mean +/- SEM, n = 6). Ketoconazole was without effect on the 3 beta-HSD-I and 17 beta-HSD activities when using [3H] pregnenolone and [3H] oestradiol, respectively, as substrates. Since ketoconazole is known to inhibit cytochrome P-450-dependent enzyme reactions, the results of the present study support the contention that cytochrome P-450 is involved in the aromatisation process.     

Urinary 5-ene-steroid excretion in non-classical congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD)

The diagnosis of non-classical 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD) is made either on the basis of significantly elevated serum levels of basal and post-ACTH 5-ene-steroids or by the presence of elevated urinary 5-ene-steroid metabolites. There has been only one report to date describing a single patient where the diagnosis was based on both serum and urinary 5-ene-steroid levels. We, therefore, measured both serum 5-ene-steroid responses to ACTH 1-24 (by RIA) and urinary 5-ene-steroid metabolites (GC-MS) in 42 hirsute premenopausal women. While the serum 5-ene-steroid profile was consistent with NC-3BHSD in 5 women, only 2 of them had increased excretion of 5-ene-steroid metabolites. Elevated 5-ene-steroid excretion was also observed in several patients with normal serum 5-ene-steroids. Detection of NC-3BHSD by either elevated serum 5-ene-steroids or increased urinary excretion of their metabolites in isolation may not therefore be reliable.     

Concerning the subcellular distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the rat adrenal.

The distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the subcellular fractions of rat adrenal gland has been determined. Based on the total activity found, 55% was in the microsomal fraction, 10% in the heavy-mitochondrial fraction, 3% in the light-mitochondrial fraction and 26% in the fraction consisting of cell-debris plus nuclei. Ninety-five percent of the total activity was recovered in the fractions. Approximately half the activity in the heavy-mitochondrial fraction could be accounted for by microsomal contamination.     

Distribution of 3 beta-hydroxysteroid dehydrogenase during development of the rat adrenal cortex and capsule

Fibroblasts of the adult adrenal cortex are considered to be nonsteroidogenic connective-tissue cells. However, it has been reported that in response to regenerative stimuli, adrenocorticotropic hormone (ACTH), and transformation to malignancy, these cells acquire characteristics of parenchymal cells, which includes delta 5, 3 beta-hydroxysteroid-dehydrogenase (delta 5, 3 beta-HSD) activity. To determine whether such delta 5, 3 beta-HSD activity in adult adrenocortical fibroblasts was due to the activation or augmentation of gene expression normally occurring during embryogenesis, a histochemical study of adrenocortical development, with particular attention to the connective-tissue capsule, was undertaken. Cryostat sections of rat embryos, from 14-days postconception (PC) to birth, and of adrenal glands 1-8, 44 and 90 days after birth were tested histochemically for delta 5, 3 beta-HSD. The same or adjacent sections were stained for PAS-positive material and reticulin, and with hematoxylin and eosin. delta 5, 3 beta-HSD activity overlapped with fibroblast-like cells and with extracellular connective-tissue components in the periphery of the glands from day-17 PC onward. delta 5, 3 beta-HSD activity over the capsule diminished shortly after birth and was absent in the adult. Appropriate controls showed that the staining within the capsule was specific and not an artifact. 3 beta-HSD activity in the capsule was more intensive when dehydroepiandrosterone (DHEA) was replaced by etiocholan-3 beta-ol-17-one (ETIO) as the steroid substrate. Furthermore, the spatial distribution of 3 beta-HSD activity in the cortex differed depending on the substrate used, and the distribution patterns changed with developmental age. (ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of peroxidase and delta 5-3 beta-hydroxysteroid dehydrogenase in the ovary of LH-treated cyclic female rats

Rats were treated with LH at 08:00 h on the first day of dioestrus or on Days 1 and 2 of dioestrus. Peroxidase activity increased within 3 h in females injected with LH on Day 1 and was associated with a depletion of ascorbate that lasted until the afternoon of Day 1. Values of both substances then returned to basal values by the morning of Day 2. A second LH injection on Day 2 produced effects similar to those seen after LH on Day 1. Females treated with LH did not display greater delta 5-3 beta-HSD activity than did controls on Day 1 after one LH injection, but did on Day 2 after two LH injections. Nonetheless, the changes were only modest by comparison with those of peroxidase. The peroxidase-ascorbate system appears to be involved in the mechanism responsible for the increased secretion of ovarian progesterone resulting from LH stimulation.     

Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.