Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Hydroxysteroids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 559 records · Page 31Linked to original sources
PubMed1977

Multihormonal regulation of sex-dependent 17beta-hydroxysteroid dehydrogenase activity in rat kidney cytosol.

The sexually differentiated activity of rat kidney cytoplasmic 17beta-hydroxysteroid dehydrogenase is subject to multihormonal regulation. Gonadectomy as well as the treatment of intact male animals with estradiol removes the repressive influence of testicular androgens, thereby allowing the activity in the male animal to reach the higher female level. The maintenance of this sexually undifferentiated activity level is a function of the thyroid gland, the adrenals and of growth hormone (somatotropin) and prolactin. Removal of the thyroid or the adrenals from intact animals decreases the activity and eliminates the sexual difference. Following the substitution of hypophysectomized animals with different hypophyseal hormones or gonadotropin preparations, only growth hormone and prolactin are effective in causing an increase in the very low activity. Thus, the sexual differentiation of renal 17beta-hydroxysteroid dehydrogenase activity observed in intact rats is clearly the result of the repressive action of testicular androgens on a multi-hormonally controlled activity level.

Adrenal Cortex↗
PubMedDate not supplied

Properties of an antiserum against Streptomyces hydrogenans 20beta-hydroxysteroid dehydrogenase.

Antiserum against crystallized 20beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was used for different immunodiffusion and immunoprecipitation tests to quantify the bacterial enzyme in cell-free supernatants of the microorganism. After immunoprecipitation and gel electrophoresis the molecular weight of the subunits of 20beta-hydroxysteroid dehydrogenase was calculated to be 27 300 +/- 700.

Antigen-Antibody Complex↗
PubMed1981

Sex-specific action of antiandrogens on androgen induced changes in hepatic microsomal 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase activity in the rat.

The ability of two antiandrogens, cyproterone acetate and flutamide, to block the induction of microsomal 3 beta-hydroxysteroid dehydrogenase and repression of microsomal 5 alpha-reductase in rat liver following administration of 5 alpha-dihydrotestosterone was investigated in male and female castrated and female gonad-intact rats. Although both antiandrogens blocked the effects of 5 alpha-dihydrotestosterone on the seminal vesicles and uteri of gonadectomized rats at the doses employed, cyproterone acetate showed no antiandrogenic activity against the two enzyme activities. On the contrary when cyproterone acetate was administered alone it elicited a response similar to that seen after androgen administration; when given simultaneously with 5 alpha-dihydrotestosterone, the induction of 3 beta-hydroxysteroid dehydrogenase activity was greater than after either steroid alone. In contrast the non-steroidal antiandrogen, flutamide, which had no intrinsic effect on either enzyme activity, effectively blocked 5 alpha-dihydrotestosterone mediated changes in the enzyme activities of female rats regardless of gonadal status. However the influence of 5 alpha-dihydrotestosterone administration on these enzyme activities in male rats was not prevented.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome.

Six types of human 17beta-hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The peroxisomal 17beta-hydroxysteroid dehydrogenase type 4 (17beta-HSD 4, gene name HSD17B4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The highest levels of 17beta-HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. A 3 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-HSDs. The N-terminal domain of 17beta-HSD 4 reveals only 25% amino acid similarity with the other types of 17beta-HSDs. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with D-3-hydroxyacyl-coenzyme A (CoA). The enzyme is not active with L-stereoisomers. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The HSD17B4 gene is stimulated by progesterone, and ligands of PPARalpha (peroxisomal proliferator activated receptor alpha) such as clofibrate, and is down-regulated by phorbol esters. Mutations in the HSD17B4 lead to a fatal form of Zellweger syndrome.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase.

Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent. These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Intracellular regulation of 17 beta-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells.

There is growing evidence that various isoforms of 17 beta-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17 beta-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the delta 4 or delta 5 pathway is limited.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

Studies of the mechanism of the increased biosynthesis of cholestanol in cerebrotendinous xanthomatosis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase.

It was recently proposed that the increased biosynthesis of cholestanol in cerebrotendinous xanthomatosis (CTX) is due to increased activity of the delta 5-3 beta-hydroxysteroid dehydrogenase involved in bile acid biosynthesis, causing increased conversion of cholesterol into cholestanol through 4-cholesten-3-one. Our attempts to confirm this hypothesis have failed. Liver biopsy specimens from two patients with CTX did not have any increased capacity to catalyze conversion of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-4-cholesten-3-one. Further, we did not find any changes in the activity of liver microsomal delta 5-3 beta-hydroxysteroid dehydrogenase after feeding rabbits with cholestanol or cholesterol. The findings are discussed in relation to our hypothesis that the accelerated biosynthesis of cholestanol in CTX is due to an increased conversion of early bile acid intermediates into cholestanol.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Regulation of oestrogen action: role of 17 beta-hydroxysteroid dehydrogenases.

The target cell responses to steroid hormones, such as oestrogens, are dependent on the expression of their receptors. Apart from receptor concentration, another key regulatory factor in steroid hormone action is the intracellular hormone concentration, which is affected by three main variables: the concentration of the steroid in plasma, local production and local conversion into metabolites. During the reproductive years the main source of oestrogens is the ovarian follicle, but in postmenopausal women most of the oestrogens are formed in peripheral tissues. The present overview deals with the formation of active oestrogens in steroidogenic tissues and in oestrogen target tissues, and the main focus is on 17 beta-hydroxysteroid dehydrogenases, which catalyse the interconversion between oestradiol and oestrone. It is evident that different 17 beta-hydroxysteroid dehydrogenase isoenzymes are responsible for the oxidation/reduction of oestradiol or oestrone in oestrogen target cells. Because these enzymes are involved in the biosynthesis and metabolism of oestrogens, they have an important physiological significance for the growth of oestrogen-dependent tissues and, hence, the growth and progression of hormone-dependent tumours.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.

20-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

[Proceedings: Male pseudohermaphroditism due to testicular 17 beta hydroxysteroid deshydrogenase deficiency (author's transl)].

A new case of 17 beta hydroxysteroid deshydrogenase deficiency is reported. In venous blood, dehydroepiandrosterone (DHA) level is normal, but the level of basal testosterone (T) is low, and delta 4 androstenedione (delta 4 A) is ten times higher than the normal level. The increase of delta4AT is characteristic after chorionic gonadotropin stimulation and over more in spermatic venous blood. Estrone (E1) produced from delta 4 A is high but estradiol (E2) level is equally low in systemic circulation and spermatic venous blood. This increase of plasmatic E2 is the witness of disturbance of E1 conversion into E2 as well as delta 4 conversion into T. It is an explantation of the lack of gynecomastia in this case. Serum gonadotropins LH and FSH are increased. This report contrast with other cases of 17 beta hydroxysteroid deshydrogenase deficiency and with the testicular feminization in which E2 and FSH levels are normal. This underline the importance of T in the LH regulation and the one of E2 in FSH control.

Adolescent↗
PubMed1992

[Partial 3-beta-hydroxysteroid dehydrogenase deficiencies].

An overview of the partial 3 beta-hydroxysteroid dehydrogenase deficiency is presented. The classical, congenital or early postnatal form is characterized by a salt-losing syndrome and/or ambiguous genitalia. The late-onset forms, only recognized for the last ten years and diagnosed with an increasing frequency, are to be systematically suspected in the presence of clinical hyperandrogenism with or without oligomenorrhea. This deficit, involved in both adrenal and gonadal tissues, seems to be transmitted by an autosomal recessive gene. An ovarian 3 beta-hydroxysteroid dehydrogenase deficit can be a primary cause of some cases of polycystic ovary syndrome and the relations with this affection are disputed. The increased ratios of delta 5 steroids/delta 4 steroids ensure the diagnostic conviction while the elevated ratio of 17-hydroxy-pregnenolone/17-hydroxyprogesterone and the normal ratio of 11-desoxycortisol/cortisol allow to eliminate the possibility of a 21-hydroxylase or 11 beta-hydroxylase deficiency, respectively. The treatment is based above all on the glucocorticoid utilization, which can lead to the return of menses and the ovulatory function, but the cutaneous symptoms of hyperandrogenism will be better controlled by cyproterone acetate out of situations of stress.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Expression of liver-specific member of the 3 beta-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity.

We have recently characterized two types of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3 beta-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3 beta-HSD protein does not display oxidative activity for the classical substrates of 3 beta-HSD, in contrast to the type I 3 beta-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5 alpha-androstane-3,17-dione (A-dione) and 5 alpha-dihydrotestosterone (DHT) to the corresponding 3 beta-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with Km values of 5.05 and 6.16 microM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with Km values of 0.12 and 1.18 microM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed2005

[Isolation and identification of Comamonas testosteroni: cloning and overexpression of 3alpha-hydroxysteroid dehydrogenase in E.coli].

OBJECTIVE: To isolate and identify 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) producing Comamonas testosteroni from soil, and to clone and overexpress 3alpha-HSD in E.coli. METHODS: Samples of pond mud were inoculated into cultural medium with androsterone as sole carbon source. The primary identification was performed according to the morphological observation, biochemical reaction and cultural characterization. To further identify the bacteria, a couple of primers were designed according to the 3alpha-HSD gene of Comamonas testosteroni. An 800 bp fragment containing 3alpha-HSD gene was obtained by PCR amplification. Then the PCR products were inserted into plasmids pET-15b to construct recombinant plasmids pET-15b. Afterwards the host bacteria containing recombinant plasmids pET-15b with proper orientation grew with isopropyl-beta-D-thioglactopyranoside (IPTG) induction. RESULTS: The isolated bacteria which could use androsterone as the sole carbon source had 85% consistency with Comamonas testosteroni. After 5 hours of IPTG induction, a recombinant protein about 29 x10(3) with enzyme activity was overexpressed in the host bacteria E.coli. BL21(DE3) pLysS. This protein could catalyze the dehydrogenization reaction of androsterone (3alpha-hydroxysteroid). CONCLUSION: A strain of Gramjnegative 3alpha-HSD producing Comamonas testosteroni was isolated from pond mud, and recombinant 3alpha-HSD with enzyme activity was overexpressed in E.coli. This work laid good foundation for the purification of recombinant 3alpha-HSD by metal chelate chromatography, and also for the construction of an enzymatic cycling method to measure serum total bile acids with recombinant 3alpha-HSD as the tool enzyme.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗
PubMedDate not supplied

[Congenital adrenal hyperplasia due to blockade of 3-beta-hydroxysteroid dehydrogenase].

Five cases of congenital 3 beta-hydroxysteroid deshydrogenase deficiency in children are reported: four boys with perineal posterior hypospadias and one girl with clitoromegaly. The salt losing syndrome was clinically overt in only three patients. The main biological character was the very high level of plasma dehydroepiandrosterone (DHA) with an elevated DHA/delta 4 androstenedione ratio. The 17 alpha-OH progesterone, though in normal biosynthesis of glucocorticoids being produced beyond the enzymatic block, was raised, but this apparently paradoxical observation may assist making the diagnosis. Deficient production of testosterone was demonstrated in the prepubertal boys by absence of postnatal rise in plasma testosterone or a decreased reponse of plasma testosterone to chorionic gonadotrophin. It is concluded that deficiency of 3 beta-hydroxysteroid deshydrogenase, now easily recognizable with the use of plasma steroids radioimmunoassay, is probably less rare than was apparent with the use of urinary steroid estimations.

3-Hydroxysteroid Dehydrogenases↗
PubMed1975

17Beta-hydroxysteroid dehydrogenase activity in Streptomyces hydrogenans.

Streptomyces hydrogenans converts 17beta-hydroxyandrost-4-ene-3-one (testosterone) to androst-4-ene-3,17-dione (androstenedione) in good yields. Time-dependence of the conversion, steroid uptake and release have been studied in vivo. Steroid analysis was done by thin-layer chromatography and recrystallization to constant specific radioactivity. After sonification of the cells the postulated 17beta-hydroxysteroid dehydrogenase activity was recovered in the 105 000 g supernatant. The enzyme was enriched by gel filtration on Sephadex G-200. It required NAD+ as cofactor. Its activity could be studied photometrically, because there are no further testosterone-netabolites. If S. hydrogenans was cultured in the presence of testosterone, estradiol or 5alphaH-dihydrotestosterone, the activity of 17beta-hydroxysteroid dehydrogenase increased.

Androstenedione↗
PubMed1991

Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase which will terminate androgen action by converting 5 alpha-dihydrotestosterone to 3 alpha-androstanediol. It is identical to dihydrodiol dehydrogenase and it can function as a 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase. Its reactions are potently inhibited by the nonsteroidal anti-inflammatory drugs (NSAIDs). A cDNA (2.1 kilobases) for 3 alpha-HSD was cloned from a rat liver cDNA expression library in lambda gt11. Portions of the cDNA insert which contained an internal EcoRI site were subcloned into pGEM3, and dideoxysequencing revealed that the cDNA contains an open reading frame of 966 nucleotides which encode a protein of 322 amino acids with a monomer Mr of 37,029. The identity of this clone was confirmed by locating two tryptic peptides and two endoproteinase Lys-C peptides from purified 3 alpha-HSD within the nucleotide sequence. The amino acid sequence of rat liver 3 alpha-HSD bears no significant homology with 3 beta-, 17 beta- or 11 beta-hydroxysteroid dehydrogenases but has striking homology with bovine lung prostaglandin F synthase (69% homology at the amino acid level and 74% homology at the nucleotide level) which is a member of the aldehyde/aldose reductase family. This sequence homology supports previous correlates which suggest that in rat 3 alpha-HSD may represent an important target for NSAIDs. The nucleotide sequence also contains three peptides that have been identified by affinity labeling with either 3 alpha-bromoacetoxyandrosterone (substrate analog) or 11 alpha-bromoacetoxyprogesterone (glucocorticoid analog) to comprise the active site (see accompanying article (Penning, T. M., Abrams, W. R., and Pawlowski, J. E. (1991) J. Biol. Chem. 266, 8826-8834]. The sequence data presented suggests that 3 alpha-HSD, prostaglandin F synthase, and aldehyde/aldose reductases are members of a common gene family.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Purification and characterization of a microbial, NADP-dependent bile acid 7 alpha-hydroxysteroid dehydrogenase.

A constitutively expressed 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) has been purified over 1200-fold, to apparent homogeneity, from an intestinal anaerobic bacterium. The purified protein had a subunit molecular mass of 32 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepharose CL-6B gel filtration gave a native molecular mass estimate of 124 kDa, suggesting that this enzyme existed as a tetramer of identical subunits. Sulfhydryl reactive compounds were potent inhibitors of 7 alpha-HSDH activity, however, metal ion chelators had no effect upon catalytic activity. The purified enzyme was highly NADP-dependent. Bile acid substrate utilization studies revealed that the enzyme was specific for the oxidation of an unhindered 7 alpha-hydroxyl group. A wide variety of bile acids and analogs were used as substrates including glycine and taurine conjugates, and methyl esters, amines, and bile alcohols. The purified 7 alpha-HSDH obeyed Michaelis-Menten kinetics. Hanes plots of substrate saturation kinetics revealed that most bile acid substrates had Km values ranging from 4 to 20 microM, while Vmax was 601 and 674 mumol/min/mg in the direction of bile acid oxidation and reduction, respectively. Primary kinetic plots and product inhibition patterns were consistent with an ordered sequential mechanism, with NADP(H) binding first. The N-terminal amino acid sequence analysis of the purified enzyme revealed a striking homology to several short, non-zinc alcohol/polyol dehydrogenases and a putative, cholate-inducible, hydroxysteroid dehydrogenase from the same organism. The high specific activity together with the stability, substrate range, and ease of purification, make this enzyme an excellent candidate for use in quantitating primary bile acids both in laboratory and clinical samples. Spectrofluorometry allowed for the quantitation of as little as 10 nM of both free and conjugated primary bile acids.

Amino Acid Sequence↗
PubMed1989

Is there any delta 5-3 beta hydroxysteroid dehydrogenase activity in preimplantation embryo of rhesus monkey?

This is the first report on the histochemical assessment of delta 5-3 beta hydroxysteroid dehydrogenase activity in all the preimplantation embryonic stages in the rhesus monkey (Macaca mulatta). An apparent stage dependent increase in enzyme activity was obtained, however, distinctively a high degree of non-specificity in enzyme reaction was noted primarily in morulae and blastocysts. Such marked non-specificity in the histochemical enzyme reaction for delta 5-3 beta hydroxysteroid dehydrogenase activity was not found in mouse blastocysts. High amounts of endogenous steroids present within rhesus embryos, or the participation of non-specific dehydrogenases could account for the observed non-specificity. Furthermore, the present report documents the pattern and degree of association (r = 0.9; P less than 0.01) between developmental stage and gestational age of preimplantation rhesus embryos, and thus provides a normal in situ cell cleavage rate of preimplantation embryo in the rhesus monkey.

3-Hydroxysteroid Dehydrogenases↗