UNIPROT:Q7LGC8 - FACTA Search

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Query: UNIPROT:Q7LGC8 (HSD)
3,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An 11 beta hydroxysteroid dehydrogenase (11 beta HSD) activity has been localized in the rat kidney by a histochemical technique which links steroid metabolism with the production of a color reaction. Oxidation of 11 beta-hydroxyandrostenedione was observed in cortical distal convoluted tubules and in medullary collecting ducts. Carbenoxolone abolished staining, no reaction was obtained with androstenedione hydroxylated at the 17 or 19 position, and oxidation of 11 beta-hydroxyandrostenedione was nicotinamide-adenine dinucleotide (NAD) dependent. These results demonstrate the presence of a dehydrogenase activity separate from the nicotinamide-adenine dinucleotide phosphate (NADP)-dependent 11 beta hydroxysteroid dehydrogenase recently purified and cloned from rat liver. We have named this activity 11 beta HSD2 to distinguish it from the NADP-dependent 11 beta HSD. Histological studies showed that 11 beta HSD2 activity does not correlate with the immunocytochemical localization of the previously defined 11 beta HSD enzyme, but rather the 11 beta HSD2 activity is localized in the distal tubules of the rat kidney. In this respect 11 beta HSD2 colocalizes with the mineralocorticoid receptor. No reaction product was obtained using cortisol or corticosterone as substrate with either NAD or NADP as cofactor. Furthermore incubation of tissue sections with 11 beta androstenedione in the presence of deoxycorticosterone completely inhibited cytochemical staining. We interpret these results as evidence of 20 reductase activity which uses the reduced cofactor at the expense of the color reaction. These results support the crucial role played by an 11 beta hydroxysteroid dehydrogenase in the local protection of type I receptors in mineralocorticoid selective tissues.
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PMID:Localization of an 11 beta hydroxysteroid dehydrogenase activity to the distal nephron. Evidence for the existence of two species of dehydrogenase in the rat kidney. 172 21

A 3 alpha-reducing activity of 5 alpha-dihydrotestosterone (5 alpha-DHT) was found in pig adrenal cytosol. The enzyme (3 alpha-hydroxysteroid dehydrogenase: 3 alpha-HSD) has been purified to homogeneity from pig adrenal cytosol by ammonium sulfate precipitation followed by DEAE-cellulose, 2', 5'-adenosine diphosphate-Sepharose and Sephadex G-100 column chromatographies. The molecular weight was estimated to be 33,000 and 39,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point was estimated to be 8.5 by isoelectric focusing. The Km and Vmax values for 5 alpha-DHT in the reduction were 10.2 microM and 10.6 nmol/min/mg. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotine amide adenine dinucleotide (NADH) in the reduction as a cofactor, but it preferentially required NADPH rather than NADH. Furthermore, the purified enzyme catalyzed not only 3 alpha-reduction of 5 alpha-DHT (9.65 nmol/min/mg), but also catalyzed 20 alpha-reduction of 17 alpha-hydroxyprogesterone (0.58 nmol/min/mg). The enzyme activity of 3 alpha-HSD was strongly inhibited by Hg2+, but it was not inhibited by medroxyprogesterone acetate and some anti-inflammatory agents. No remarkable differences was demonstrated between 3 alpha-HSD and 20 alpha-HSD activity under the influence of heat treatment, divalent cation, anti-inflammatory agents and some inhibitory steroids. These results strongly suggest that 3 alpha-HSD purified from pig adrenal cytosol is a bi-functional enzyme catalyzing 3 alpha- and 20 alpha-HSD activities.
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PMID:[Purification and some properties of 3 alpha-hydroxysteroid dehydrogenase from pig adrenal cytosol]. 180 59

Neonatal pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) catalyzed the oxidation of 20 beta-hydroxysteroids, 17 alpha,20 beta-dihydroxypregn-4-en-3-one and 20 beta-hydroxypregn-4-en-3-one in the presence of beta-nicotinamide adenine dinucleotide phosphate (beta-NADP+). The behavior of 20 beta-HSD activity toward the substrate of 17 alpha,20 beta-dihydroxypregn-4-en-3-one differed from the catalytic reaction for 20 beta-hydroxypregn-4-en-3-one. The enzyme could catalyze not only 20 beta-hydroxysteroids but also 20 alpha-hydroxy-5-ene steroids, 20 alpha-hydroxypregn-5-en-3 beta-ol and 17 alpha,20 alpha-hydroxypregn-5-en-3 beta-ol with 22.1 and 8.7% of activity relative to 20 beta-hydroxypregn-4-en-3-one, respectively. The enzyme preferentially required beta-NADP+, and also utilized beta-nicotinamide adenine dinucleotide beta-NAD+ and beta-nicotinamide adenine dinucleotide 3'-phosphate (beta-3'-NADP+) nonspecifically as the cofactor. The optimum pH was observed at pH 7.5 with the substrate of 20 beta-hydroxypregn-4-en-3-one. The activation energies obtained from oxidation-reduction reactions of 20 beta-HSD for the substrate of 20 beta-hydroxypregn-4-en-3-one, progesterone and 17 alpha-hydroxyprogesterone were estimated at 13.8, 27.0 and 20.0 kcal/mol, respectively.
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PMID:20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: reverse catalytic (oxidation) reaction. 189 1

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

A fluorometric enzymatic method for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates in human serum has been developed. A simple and fast purification and preconcentration procedure using Sep Pak C18 cartridges was employed for the UDCA extraction from human serum. UDCA and its conjugates were determined in the extracted sample by an equilibrium method based on the enzymatic conversion of the 7 alpha-hydroxy group into 7-oxo group by beta-nicotinamide adenine dinucleotide phosphate in the presence of 7 beta-hydroxysteroid dehydrogenase (7 beta-HSD) and the produced NADPH was monitored fluorometrically. The 7 beta-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC No. 27555) and purified by affinity chromatography. The method has a limit of detection of 0.8 microM in serum and the precision varied from 6.1 to 2.0% for low and high concentrations, respectively. The recovery of UDCA from serum samples was about 99% (range 85-105%). The method was successfully applied to UDCA determination in serum samples from patients treated with UDCA for primary biliary cirrhosis.
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PMID:Determination of ursodeoxycholic acid in serum by a new fluorometric enzymatic method using 7 beta-hydroxysteroid dehydrogenase from Clostridium absonum. 267 76

The aim of this study was to determine whether steroidogenesis occurs in human immature oocytes aspirated from follicles during gynecologic laparotomy. delta 5-3 beta-Hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities were detected by Dickmann and Dey's reaction medium consisting of 1.8 mg substrate (pregnenolone or 17 beta-estradiol [E2]), 4 mg nicotinamide-adenine dinucleotide, 2 mg nitro-blue tetrazolium, 10 ml 0.1 M phosphate buffer. The activity of adenylate cyclase was examined by ultrastructural-cytochemical study using 5'-adenylyl-imidodiphosphate (AMP-PNP) as a substrate in Vorbrodt's medium of 0.005 M AMP-PNP, 0.001 M MgCl2, 0.02 M SrCl2, 0.01 M NaF, 0.002 M theophylline, 0.01 M Tris-HCl. Furthermore, in indirect immunofluorescence study, the presence of endogenous progesterone and E2 and the activity of delta 5-3 beta-HSD were demonstrated. The results suggest that steroidogenesis, through the adenylate cyclase-cyclic adenosine 3':5'-monophosphate system, in human oocytes may play some important role in oocyte maturation, fertilization, and early embryonic development. The implication of steroid-producing activities of the human oocytes for cytoplasmic maturation is discussed.
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PMID:Cytochemical study of steroid-producing activities of human oocytes. 630 92

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSA) was used to affinity-label the NADH binding region of 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) to further test our hypothesis [Sweet, F., & Samant, B. R. (1980) Biochemistry 19, 978-986] that 3 alpha and 20 beta activities occur at the same active site. Incubation of 3 alpha, 20 beta-HSD (0.45 microM) with FSA (125 microM) at pH 7.0 and 0 degrees C caused simultaneous loss of 3 alpha and 20 beta activities by a first-order kinetic process, with t1/2 = 300 min for both activities. Dinucleotides and adenosine mononucleotides which acted as competitive inhibitors protected 3 alpha, 20 beta-HSD against inactivation by FSA in a concentration-dependent manner, in the order reduced nicotinamide dinucleotide phosphate greater than oxidized nicotinamide dinucleotide phosphate greater than adenosine diphosphate-ribose greater than adenosine diphosphate greater than adenosine monophosphate (AMP) greater than adenosine. Oxidized and reduced nicotinamide mononucleotides (NMH and NMNH) and steroid substrates did not protect 3 alpha, 20 beta-HSD against affinity labeling by FSA. Although NMN was not a competitive inhibitor of 3 alpha, 20 beta-HSD, NMN with AMP and also AMP with NMNH produced positive cooperativity for competitive inhibition of 3 alpha, 20 beta-HSD. The results from FSA affinity labeling of the cofactor region confirm that both 3 alpha and 20 beta activities share the same active site of 3 alpha, 20 beta-HSD and suggest a model of cofactor binding and promotion of enzyme activity. The adenosine 5'-phosphate component anchors the NAD or NADH to an adenosine domain in the cofactor binding region. The nicotinamide nucleotide component then carries out the hydrogen-transfer reaction at a neighboring domain near the steroid binding region.
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PMID:Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside. 694 73

Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extremely sensitive to SH reagents such as N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid). NEM produced time- and concentration-dependent inactivation of DD3 in a pseudo-first-order reaction manner. This inactivation was prevented by NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, 2',5'-ADP and 2'-AMP but not by substrates, NAD+, nicotinamide mononucleotide or 5'-ADP.DD3 was absorbed by an affinity column of thiopropyl-Sepharose 6B, but enzyme incubated with both NEM and NADP+ was not. Moreover, one [14C]NEM molecule was incorporated into a cysteine of DD3 in the presence, and two cysteines of DD3 in the absence, of NADP+. These results suggested that two cysteine residues were modified per enzyme molecule by NEM, one was protected by NADP+ and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the digestion with lysyl endopeptidase of [14C]NEM-modified DD3 could be separated by reverse-phase HPLC. P1, which was radiolabelled by [14C]NEM only in the absence of NADP+, showed the following sequence; H2N-Tyr-Lys-Pro-Val-Xaa-Asn-Gln-Val-Glu- NEM.Cys-His-Pro-Tyr-Phe-Asn-Gln-Ser-Lys-COOH (Xaa indicates a possible cysteine residue). This sequence was very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also highly conserved in the aldo-keto reductase superfamily. The sequence of P2, which had radioactivity in both the absence and presence of NADP+, also contained an NEM-modified cysteine and was similar in sequence to the regions located in loop A of rat 3 alpha-HSD/DD. The present study suggests that P1, which may have a cysteine residue corresponding to Cys-193 of rat 3 alpha-HSD/DD, functions in the alteration of DD3 activity depending on the modulation of NADP(+)-binding ability through a thiol/disulphide exchange reaction similar to that of rat 3 alpha-HSD/DD shown in our previous results; while P2, which may have a cysteine residue corresponding to Cys-145 of rat 3 alpha-HSD/DD, may be located near the surface of the enzyme molecule.
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PMID:The role of cysteine in the alteration of bovine liver dihydrodiol dehydrogenase 3 activity. 764 30

Mineralocorticoid (MC) receptors are found in classic (e.g., kidney) and nonclassic (e.g., heart and aorta) tissues. MC receptor specificity at either site is conferred by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which metabolizes glucocorticoids to inactive 11-ketosteroids. Because the heart and vasculature may be target tissues for aldosterone, this study was undertaken. Its objectives were to measure 11 beta-HSD at these sites and to compare levels of activity in the atria and ventricles of different species. Toward this end we first determined levels of plasma corticosterone (B) and cortisol (F) (by using radioimmunoassay), in a variety of species, for subsequent correlation with cardiac 11 beta-HSD activity. 11 beta-Dehydrogenation of glucocorticoids was then assayed in ventricles and atria as well as aorta. Tissue homogenates containing 1 to 5 mg protein were incubated for 1 hour in the presence of 1 muCi 5 x 10(-9) mol/L tritiated B or tritiated F and 5 x 10(-4) mol/L oxidized nicotinamide adenine dinucleotide phosphate. Steroid separation and quantitation were achieved by using reverse-phase high-performance liquid chromatography coupled to an online radioisotope detector. For species in which B circulates at relatively high concentrations (rat, rabbit, pig), high levels of dehydrogenation of B to 11-dehydrocorticosterone (A) were observed in both atria and ventricles. Overall, cardiac B to A conversion levels corresponded to between 0.3 and 0.4 pmol A formed/mg protein/hr. 11 beta-HSD activity was also detected in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Species diversity of 11 beta-hydroxysteroid dehydrogenase in the cardiovascular system. 779 96

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the conversion of cortisol to cortisone and plays an important role in the mammalian kidney in regulating cortisol access to the mineralocorticoid receptor. 11 beta HSD-deficient states, such as the syndrome of apparent mineralocorticoid excess (AME), and licorice ingestion result in hypertension in which cortisol acts as a mineralocorticoid. A gene and complementary DNA sequence encoding type I human 11 beta HSD have been described, but this gene is normal in patients with AME. Separate 11 beta HSD isoforms have been described in rat and rabbit kidney, but 11 beta HSD has not been characterized in human kidney. Kinetic analysis of 11 beta HSD activity in human fetal kidney microsomes revealed only a high affinity isoform (apparent Km, 60 nmol/L for cortisol, 13 nmol/L for corticosterone), the activity of which was exclusively nicotinamide adenine dinucleotide (NAD) dependent. No 11-oxo-reductase activity was seen in either renal homogenates or microsomes. 11 beta-Dehydrogenase activity was inhibited by glycyrrhetinic acid (the active ingredient in licorice) in a competitive fashion, with a Ki of 8.7 nmol/L. This 11 beta HSD isoform was clearly distinct from the type I h11 beta HSD enzyme, in that COS-1 cells transfected with type I h11 beta HSD complementary DNA expressed a low affinity (apparent Km, 2.13 mumol/L) isoform, the activity of which was NAD phosphate dependent. 11-Oxo-reductase activity was present in intact transfected cells (apparent Km for cortisone, 0.36 mumol/L), but not in cell lysates. In contrast to the cloned, low affinity, type I h11 beta HSD enzyme, human kidney contains a high affinity NAD-dependent 11 beta HSD isoform. It seems probable that this isoform is responsible for protecting the renal mineralocorticoid receptor from glucocorticoid excess, and a defect in its activity may explain AME.
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PMID:Human kidney 11 beta-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform. 804 66

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