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)

Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case of Escherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD+ or NADP+ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1 mM methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, Km for substrate and cofactors), it resembles its counterpart in E. coli K12. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8 M urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity of S. marcescens unlike in Salmonella typhimurium and E. coli K12 where it is a minor or a negligible component.
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PMID:Methionine-repressible homoserine dehydrogenase of Serratia marcescens: purification and properties. 18 74

3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.
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PMID:Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. 155 29

7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.
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PMID:Purification and characterization of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine. 348 Aug 90

The cDNA coding for pig testicular 3 alpha/beta (20 beta)-hydroxysteroid dehydrogenase was expressed in Escherichia coli by placing it under the control of an isopropylthiogalactoside (IPTG) inducible tac promoter. Production of 3 alpha/beta (20 beta)-HSD was demonstrated by Western blotting and by catalytic activity with 5 alpha-dihydrotestosterone as a substrate for 3 alpha/beta-HSD, and progesterone and 17 alpha-hydroxyprogesterone as substrates for 20 beta-HSD in the presence of NADPH. The 3 alpha/beta (20 beta)-HSD enzyme was detected in a soluble fraction of the lysate of E. coli added to IPTG to induce the synthesis of the protein. Its molecular weight was estimated to be 30.5 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant 3 alpha/beta (20 beta)-HSD was purified to apparent homogeneity as determined by SDS-PAGE by column chromatography using DEAE-cellulose. The purified enzyme reduced not only steroids but also prostaglandins and other carbonyl compounds including aldehydes, ketones and quinones as demonstrated in native enzymes purified from pig testes. The amino terminus of the purified enzyme was serine which was coded next to the ATG start codon, and the sequence of the amino terminal 24 residues was identical with the coding amino acid in the cDNA; whereas, the amino terminus of the native 3 alpha/beta (20 beta)-HSD was not detected suggesting that the N-terminal amino acid was blocked.
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PMID:Direct expression of pig testicular 3 alpha/beta (20 beta)-hydroxysteroid dehydrogenase in Escherichia coli. 757 8

The classical form of the enzyme 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3 beta-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3 beta HSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3 beta HSD I and III function as 3 beta-hydroxysteroid dehydrogenases and 5-en-->4-en isomerases using NAD+ as a cofactor. The enzymatic function of 3 beta HSD II has not been completely characterized. Mouse 3 beta HSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3 beta HSD I is expressed in the gonads and adrenal glands of the adult mouse. 3 beta HSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.
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PMID:The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function and tissue-specific expression. 762 43

Seminiferous tubules prepared from adult rats cultured for 48 h in serum-free conditions produce multiple biological factors that modulate Leydig cell steroidogenic function in vitro. Using gel filtration chromatography, it was shown that seminiferous tubular culture medium (STCM) contained at least three inhibitory activities designated AI, AII, and AIII that inhibited testosterone production by purified Leydig cells. The factor that induced AIII activity, designated Leydig cell inhibitor (LCI), was further purified to apparent homogeneity by sequential HPLC using gel permeation, C8-, C18-, C2/C18-reversed-phase, and microbore anion exchange columns. When this batch of purified factor was resolved by SDS-PAGE under reducing conditions, only a single silver stained band with an apparent M(r) of 21,000 was detected. Protein sequence analysis using about 100 pmol of purified LCI revealed that its N-terminus was blocked. Incubation of this highly purified factor with Percoll gradient purified Leydig cells induced a dose-dependent inhibition of hCG-stimulated testosterone production. LCI inhibited the basal testosterone production and hCG-stimulated cAMP production by Leydig cell dose-dependently. It also inhibited the forskolin- and cholera toxin-stimulated testosterone and cAMP production but had no apparent effect on the binding of 125I-labeled hCG to LH receptors. These data suggest that this LCI exerts its inhibitory action at steps beyond the LH receptors but prior to the cAMP formation by affecting the adenylate cyclase activity directly or indirectly through inhibition of the stimulatory G-protein (Gs-protein); however, it is also possible that it decreases the coupling of the receptors to the Gs-protein. LCI also inhibited the conversion of exogenously added 22R-hydroxycholesterol, pregnenolone, progesterone, and 17 alpha-hydroxyprogesterone to testosterone. However, it had no effect on the conversion of dehydroepiandrostenedione and androstenedione to testosterone. These data strongly suggest that LCI affects the steroidogenic enzymes metabolizing cholesterol to testosterone, the cytochrome P-450 side-chain cleavage (P-450SCC), and cytochrome P-450 17 alpha-hydroxylase/17,20-lyase (P-450C17). However, it has no effect on the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) enzyme activities. Based on the results of the present study, it is apparent that this LCI is distinct from other known potent Leydig cells inhibitors such as interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta). The LCI appears to involve in the paracrine regulation of Leydig cell function.
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PMID:Rat seminiferous tubular culture medium contains a biological factor that inhibits Leydig cell steroidogenesis: its purification and mechanism of action. 798 48

NADPH-dependent 3 alpha/beta-hydroxysteroid dehydrogenase (3 alpha/beta-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. The molecular weight of the enzyme was estimated to be 31 kDa by SDS-polyacrylamide gel electrophoresis and 40 kDa by gel filtration chromatography indicating that the native 3 alpha/beta-HSD is a monomer. The isoelectric point of the purified enzyme was found to be 6.2 by density gradient isoelectric focusing and 6.4 by chromatofocusing. The enzyme reduced both 5 alpha- and 5 beta-DHT, 5 alpha- and 5 beta-dihydroprogesterone, 5 alpha- and 5 beta-dihydrocortisol, prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2 and 13,14-dihydro-15-keto-prostaglandin F2 alpha. Moreover, the enzyme caused rapid reduction of other carbonyl compounds including aldehydes, ketones and quinones. The rates of reduction of these compounds are fast relative to the rates of reduction of steroids and prostaglandins. The purified enzyme was inhibited by AgNO3, SH-reagent, quercetin, hexesterol, stilbestrol, disulfiram and divalent cation such as Cu2+, Hg2+ and Cd2+. The two enzymes show certain similarities (e.g. molecular weight, cross-reactivity to a common antibody) and certain striking differences (e.g. pI, effects of various inhibitors and greater enzyme activity towards steroids (neonatal form) or prostaglandins (mature form). Reasons are give for suggesting that these enzymes are closely related to carbonyl reductase.
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PMID:Purification and characterization of 3 alpha/beta-hydroxysteroid dehydrogenase from mature porcine testicular cytosol. 814 2

A bacterial strain, B-0831, which produced 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) was isolated and identified as belonging to the genus, Pseudomonas. Molecular weights of the purified 3 alpha HSD, determined by SDS-PAGE and by chromatography on Sephacryl S-200, were about 25 and 50 kDa, respectively. A genomic library of Pseudomonas sp. B-0831, prepared in the plasmid vector pACYC184, was screened with probes based on the amino acid (aa) sequence of the protein to obtain the plasmid, p3 alpha HSD1, identified by hybridization with the probes, that contained a 2.4-kb insert from Pseudomonas DNA. When the 1.4-kb SphI fragment of p3 alpha HSD1 was inserted into the vector, pUC118, and introduced into Escherichia coli DH1 under the control of lacZ promoter in the vector, the transformants produced 200-fold more 3 alpha HSD intracellularly than Pseudomonas sp. B-0831. Sequence analysis of the 3 alpha HSD gene revealed that an ORF encoding 3 alpha HSD consists of 254 aa, with a calculated M(r) of 25,761, suggesting that the enzyme consists of homodimer subunits.
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PMID:Cloning and expression of a Pseudomonas 3 alpha-hydroxysteroid dehydrogenase-encoding gene in Escherichia coli. 834 21

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum.
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PMID:Escherichia coli flavodoxin sepharose as an affinity resin for cytochromes P450 and use to identify a putative cytochrome P450c17/3beta-hydroxysteroid dehydrogenase interaction. 934 69

While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.
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PMID:Purification and characterization of hamster liver microsomal 7alpha-hydroxycholesterol dehydrogenase. Similarity to type I 11beta-hydroxysteroid dehydrogenase. 963 80


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