Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.1.1.3 (HSD)
 3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetic behavior of homogeneous rat liver 11 beta-hydroxysteroid dehydrogenase (11-HSD) was investigated. The purified enzyme catalyzed oxidation of the 11 beta-hydroxy steroids, cortisol and corticosterone, to their 11-oxo products. The reverse 11-oxoreductase was not detected. Initial velocity studies of 11 beta-dehydrogenase were consistent with a sequential bireactant mechanism. Glycyrrhetinic acid, a competitive inhibitor of corticosterone oxidation, was uncompetitive with respect to NADP+. The observed inhibition patterns were consistent with an ordered sequential mechanism with NADP+ adding to the enzyme first. Analogs of NADP+ and NAD+ did not inhibit steroid oxidation by 11-HSD, nor did the products of the 11 beta-dehydrogenase reaction slow oxidation, or catalyze reduction. Ligand binding studies generated patterns that supported the ordered sequential mechanism derived from kinetic studies. The kinetic behavior of 11-HSD is therefore similar to other alcohol dehydrogenases. The basis for the apparent inability of homogeneous 11-HSD to catalyze reduction remains to be established.
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PMID:Kinetic studies on rat liver 11 beta-hydroxysteroid dehydrogenase. 195 1

We have recently demonstrated that treatment of pregnant baboons with androstenedione (delta 4 A) at midgestation to increase estrogen production induced a pattern of placental cortisol (F) metabolism which was similar to that at term and resulted in de novo F production by the fetus, presumably by activation of the fetal hypothalamic-pituitary-adrenocortical axis. The present study was designed to examine the subcellular events in the fetal adrenal that were apparently stimulated by estrogen-induced alterations in transplacental corticosteroid metabolism. Therefore, we determined the effects of estrogen treatment at midgestation and removal of estrogen action near term on the specific activity of the rate-limiting enzymes delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17-hydroxylase-17,20-lyase (17 alpha-OHase). Fetal adrenals were obtained on day 100 (n = 11) or day 165 (n = 11) of gestation (term = day 184) from untreated animals, on day 100 from animals receiving delta 4 A daily between days 70-100 (n = 9) to increase placental estrogen production, and on day 165 from baboons treated daily between days 130-164 with antiestrogen ethamoxytriphetol (MER-25; n = 7). The activity of 17 alpha-OHase was determined by incubating adrenal microsomes (105,000 x g) with [3H] progesterone, NAD+, and NADH in phosphate buffer. The radiolabeled products 17-hydroxyprogesterone, delta 4 A, and testosterone were purified, and enzyme activity expressed as picograms of product per min/mg tissue. The activity of 3 beta HSD was determined by incubating adrenal microsomes with [3H]pregnenolone and NAD+ in phosphate buffer. The radiolabeled progesterone product was purified, and enzyme activity was expressed as nanograms per min/mg tissue. Treatment with delta 4 A increased estrogen concentration at midgestation 3-fold to levels comparable to those measured near term. Although fetal adrenal weight was greater at term than at midgestation (p less than 0.05), weight was not increased by delta 4 A treatment. The specific activity (mean +/- SE) of fetal adrenal 17 alpha-OHase at midgestation (181 +/- 29) was increased (P less than 0.05) 3-fold by treatment with delta 4 A to levels (591 +/- 105) comparable to those in adrenal microsomes prepared from untreated animals near term (816 +/- 130). Enzyme activity in adrenals of MER-25-treated baboons was 40%, but not significantly lower than that in term controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: adrenal delta 5-3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase-17,20-lyase activity. 201 57

We have previously shown that the change in transuteroplacental cortisol (F)-cortisone (E) metabolism in vivo from preferential reduction (E to F) at midgestation to oxidation by term (F to E) does not occur in baboons in which the production or action of estrogen have been blocked. Moreover, because the administration of androstenedione (delta 4A) to baboons increased estradiol (E2) production at midgestation and induced a pattern of F-E metabolism similar to that at term, we suggested that estrogen regulates placental F-E interconversion. The present study was designed to ascertain whether estrogen regulates the activity of the placental 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) enzyme catalyzing the oxidation of F to E. Placentas were obtained on day 100 (n = 10) and day 165 (n = 10) of gestation (term = day 184) from untreated baboons, on day 100 from animals (n = 7) treated with delta 4A between days 70-100 of gestation, and on day 165 from animals in which placental estrogen was decreased by fetectomy (n = 5) on day 100 of gestation. Tissue was homogenized in phosphate buffer (pH 7.4) and microsomal fractions (105,000 x g) incubated (37 C; 2 min) in buffer containing 2.7 mM NAD+ and 0.03-1.0 microM [3H]F. Serum concentrations of E2 (nanograms per ml) in untreated baboons on day 100 (0.7 +/- 0.2) were 3-fold lower than those at term, increased (P less than 0.05) by delta 4A treatment (2.4 +/- 0.3), and decreased (0.12 +/- 0.01; P less than 0.05) by fetectomy. The specific activity (picomoles of E per min/mg protein) of placental 11 beta HSD in untreated baboons at midgestation (134 +/- 17) was increased (P less than 0.01) 3-fold by delta 4A treatment. Enzyme activity at term (148 +/- 29) was similar to that at midgestation, but markedly decreased (P less than 0.01) by fetectomy (16 +/- 4). Placental capacity to oxidize F to E (micromoles per min/placenta) in untreated baboons was 3-fold greater (P less than 0.01) at term (88 +/- 15) than at midgestation and was markedly reduced (P less than 0.01) by fetectomy (3 +/- 1). Collectively, these findings indicate that the activity of the placental 11 beta HSD enzyme catalyzing the oxidation of F to E is increased in baboons in which placental estrogen production was elevated at midgestation and decreased in animals when estrogen formation was inhibited by fetectomy.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of 11 beta-hydroxysteroid dehydrogenase activity in the baboon placenta by estrogen. 232 4

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.
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PMID:Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase. 261 66

Two non-steroidal mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver have been synthesized: 1-(4'-nitrophenyl)-2-propen-1-ol (I), and 1-(4'-nitrophenyl)-2-propyn-1-ol (II). Both of these compounds inactivate homogeneous 3 alpha-HSD in a time- and concentration-dependent manner only in the presence of NAD+. Analysis of the pseudo-first-order inactivation data gave a Kd of 1.2 mM for the allylic alcohol and a t1/2 (time required to promote a 50% loss of enzyme activity) for the enzyme of less than 10 s at saturation. Similar inactivation studies with the acetylenic alcohol gave a Kd of 1.5 mM and a t1/2 for the enzyme of 9.9 min at saturation. The allylic alcohol and acetylenic alcohol are oxidized stereoselectively by the enzyme, yielding a Km of 2.0 mM and a Vmax. of 0.58 mumol/min per mg for the allylic alcohol and a Km of 0.75 mM and a Vmax. of 0.29 mumol/min per mg for the acetylenic alcohol. Effective partition ratios (kcat./kinact.) are low for both alcohols: for the allylic alcohol, 5.3; and for the acetylenic alcohol, 141. H.p.l.c. indicates that the Michael acceptors 1-(4'-nitrophenyl)-2-propen-1-one (III) and 1-(4'-nitrophenyl-2-propyn-1-one (IV) are the products of the enzymic oxidation of the corresponding alcohols. The latter compound (IV) was trapped as its monothioether adducts before h.p.l.c. analysis. The Michael acceptors III and IV inactivate the 3 alpha-HSD in the absence of NAD+ at a rate too high to accurately measure and titrate the enzyme in a stoichiometric manner. Enzyme inactivated by I and NAD+, II and NAD+, III or IV is not re-activated by gel filtration or dialysis, implying a stable covalent bond has been formed between the enzyme and the inactivators. A screen of five other HSDs, and two aliphatic alcohol dehydrogenases, indicates that alcohol I is a selective inactivator of rat liver 3 alpha-HSD. It is concluded that 3 alpha-HSD generates non-steroidal alkylating agents (III and IV) that potently inactivate the enzyme with low effective partition coefficients. This report of non-steroidal mechanism-based inactivators of 3 alpha-HSD may provide a precedent for the development of related compounds to act as suicide substrates of other HSDs.
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PMID:Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase. 264 May 66

The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites.
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PMID:Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids. 299 30

A high-performance liquid chromatographic method for the simultaneous determination of individual sulphated 3 alpha- and beta-hydroxysteroids in serum using 3 alpha- and beta-hydroxysteroid dehydrogenases (3 alpha-HSD and beta-HSD, respectively) immobilized on one column and a fluorimeter to detect the resulting NAD+ to NADH transformation is described. Individual sulphated 3 alpha- and beta-hydroxysteroids in serum are extracted with ethanol, solvolysed with sulphuric acid in ethyl acetate and then separated by high-performance liquid chromatography. The hydroxysteroids thus separated are subsequently mixed with NAD+ and then passed through the column in which the following catalytic reaction occurs: (formula, see text) The detection limits are as low as 0.5-1.0 microgram/dl for sulphated 3 alpha- or beta-hydroxysteroids in serum. The present assay method is highly specific, reliable and reproducible and is thus applicable to a clinical study on the metabolism of sulphated 3 alpha- and beta-hydroxysteroids in patients with adrenal or gonadal diseases.
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PMID:Simultaneous assay for individual sulphated 3 alpha- and beta-hydroxysteroids in serum using high-performance liquid chromatography combined with 3 alpha- and beta-hydroxysteroid dehydrogenases immobilized on one column. 322 Sep 14

Recently, a protein fraction [follicle regulatory protein (FRP)] which inhibits FSH-induced granulosa cell aromatase activity was isolated from both human and porcine follicular fluid. In this study, the actions of FRP on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity were examined using granulosa cells obtained from hyperstimulated patients undergoing oocyte aspiration for in vitro fertilization. Granulosa cells were cultured with 0, 167, or 500 micrograms/ml FRP with or without human menopausal gonadotropin (hMG; 10 mIU/ml). After 48 h, the medium (S) was removed and stored. Cells then were mechanically lysed and centrifuged at 10,000 X g. The supernatant was further centrifuged (100,000 X g) to obtain a microsomal fraction (M) and cytosol (C). The M fraction was resuspended in medium 199 with 10(-6) M pregnenolone plus 5 microM NAD+ and incubated for 2 h to determine 3 beta-o1 dehydrogenase activity. The S, C, and M fractions were all assayed for progesterone (P) by RIA. hMG markedly increased P concentrations in the S and C fractions. The M fraction demonstrated a hMG-dependent enhancement in 3 beta-HSDH activity. However, the hMG-associated S, C, and M P levels were decreased in granulosa cells coincubated with FRP. In conclusion, ovarian steroidogenesis may be dependent on the integrated interactions of both gonadotropins and local nonsteroidal paracrine/autocrine modulators of granulosa cell function.
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PMID:Effect of human menopausal gonadotropin and follicle regulatory protein(s) on 3 beta-hydroxysteroid dehydrogenase in human granulosa cells. 392 17

The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.
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PMID:Characterization of ligand-induced states of maize homoserine dehydrogenase. 635 97

A partial characterization of human term placental 3 beta-HSDH in mitochondria is reported. Apparent KM of pregnenolone: 70 nM. A dose-dependent stimulation of 3 beta-HSDH by NAD+ or NADP+ was observed in the range from 10(-6) to 10(-3) M (KM value of NAD+: 20 microM). At equimolar concentrations NAD+ is more than 10-fold as effective a cofactor of the 3 beta-HSDH than NADP+. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40-45 degrees C. A rapid loss of 3 beta-HSDH activity was found after preincubation of the enzyme at 37 degrees C after 30 min; less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg2+, Ca2+ Sr2+ and Ba2+ (1-100 mM). A strong inhibition was achieved with 1 mM Zn2+, Cd2+, Cu2+ and 10 mM and 100 mM Fe2+, Mn2+, Co2+ and Ni2+.
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PMID:Partial characterization of placental 3 beta-hydroxysteroid dehydrogenase (EC 1.1.1.145), delta 4-5isomerase (EC 5.3.3.1) in human term placental mitochondria. 695 97


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