Separation of a purified adrenal 20α-hydroxysteroid dehydrogenase

Matthijssen, Charles; Mandel, Joyce E.; Seiden, P.T.

doi:10.1016/0926-6569(64)90229-9

Cited by 15 publications

(6 citation statements)

References 5 publications

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“…20a-HSD activity has been reported in various steroidproducing tissues such as ovary, testis, adrenal, uterus and placenta [1,[8][9][10][11]]. Since progesterone is the precursor of biologically active steroids such as androgen, oestrogen and corticoids, 20a-HSD may be involved in the regulation of biosynthesis of these hormones.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning of cDNA for rat ovarian 20 α-hydroxysteroid dehydrogenase (HSD1)

Miura

Shiota

Noda

et al. 1994

Biochemical Journal

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20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD, EC 1.1.1.149) catalyses the conversion of progesterone into 20 alpha-dihydroprogesterone (20 alpha-OHP). Previously, we purified the enzyme (37 kDa) from rat ovary and determined its N-terminal amino acid sequence. In the present study we succeeded in cloning a full-length 20 alpha-HSD cDNA. mRNA was extracted from immature rat ovaries after successive treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A cDNA library was constructed in lambda ZAP. For screening, a 576 bp probe was amplified by the PCR using mixed primers based on the N-terminal sequence of 20 alpha-HSD, and labelled with [32P]dCTP. Eight positive clones were isolated from 1.2 x 10(4) recombinants. Analysis of the nucleotide sequence revealed that one clone of 1.2 kbp cDNA (pHSD12-07) contained a polyadenylation site and an open reading frame encoding 323 amino acids with the N-terminal sequence of 20 alpha-HSD. The fusion protein of pHSD12-07 produced by Escherichia coli reacted with a specific polyclonal antibody generated against rat ovarian 20 alpha-HSD. In addition, the in vitro transcription-translation product produced by Xenopus oocytes showed 20 alpha-HSD activity and Northern-blotting analysis revealed that the ovaries from normal adult rats contained a 1.2 kb mRNA. Thus we succeeded in isolating a clone encoding the full length of rat ovarian 20 alpha-HSD. The sequence showed high similarity with those of rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), bovine lung prostaglandin F synthase (PGFS), human liver chlordecone reductase (CDR), frog lens rho-crystallin and aldose reductases, indicating that 20 alpha-HSD belongs to the aldo-keto reductase family.

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Section: Introductionmentioning

confidence: 99%

Molecular cloning of cDNA for rat ovarian 20 α-hydroxysteroid dehydrogenase (HSD1)

Miura

Shiota

Noda

et al. 1994

Biochemical Journal

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“…It has been suggested that the enzyme is a T-cell differentiation marker (Ihle et al 1981). In addition to being present in thymic-derived lymphocytes, 20 -HSD activity has been found in many steroidogenic tissues such as the liver, ovary, testis, adrenal and placenta (Wiest 1959, Matthijssen et al 1964, Armstrong & King 1971, Pineda et al 1985, Nakajin et al 1989 and also in haemopoietic cells, erythrocytes and certain micro-organisms (Weinstein 1977, Sharaf & Sweet 1982, Hapel et al 1985, Rimsay et al 1988. While 20 -HSD has been shown to play a major role in the termination of pregnancy in the rat and rabbit, the role of 20 -HSD in the human is still ill-defined.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a human 20alpha-hydroxysteroid dehydrogenase

Zhang

Dufort²,

Rheault³

et al. 2000

Journal of Molecular Endocrinology

100

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It has been suggested that 20 -hydroxysteroid dehydrogenase (20 -HSD) is a T-cell differentiation marker in mice. In the human, this enzyme has generally been associated with types 1 and 2 17 -HSDs, which belong to the short-chain alcohol dehydrogenase family, whereas the rat, rabbit, pig and bovine 20 -HSDs are members of the aldoketo reductase superfamily, which also includes the 3 -HSD family. In this study, we report the cloning, from a human skin cDNA library, of a cDNA that shows, after transfection into human embryonic kidney (HEK-293) cells, high 20 -HSD activity but negligible 3 -and 17 -hydroxysteroid dehydrogenase activities. A comparison of the amino acid sequence of the human 20 -HSD with those of other related 20 -and 3 -HSDs indicates that the human 20 -HSD shares 79·9, 68·7 and 52·3% identity with rabbit, rat and bovine 20 -HSDs, whereas it shows 97, 84 and 65% identity with human type 3, type 1 and rat 3 -HSDs. In contrast, the enzyme shares only 15·2 and 15·0% identity with type 1 and type 2 human 17 -HSDs. DNA analysis predicts a protein of 323 amino acids, with a calculated molecular weight of 36 767 Da. In intact transfected cells, the human 20 -HSD preferentially catalyzes the reduction of progesterone to 20 -hydroxyprogesterone with a K m value of 0·6 µM, the reverse reaction (oxidation) being negligible. In a cell cytosolic preparation, the enzyme could use both NADPH and NADH as cofactors, but NADPH, which gave 4-fold lower K m values, was preferred. We detected the expression of 20 -HSD mRNA in liver, prostate, testis, adrenal, brain, uterus and mammary-gland tissues and in human keratinocyte (HaCaT) cells. The present study clearly indicates that the genuine human 20 -HSD belongs to the aldoketo reductase family, like the 20 -HSDs from other species.

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“…In vertebrate species, 20a-HSDH is thought to be a key enzyme involved in tissue-specific regulation of steroid hormone metabolism (10, 39). Various forms of 20a-HSDH are found in the major steroid-producing tissues of mammals (adrenals, ovaries, testes, and placenta) and in liver, kidney, muscle, lymphatic organs, fibroblasts, and hematopoietic cells (4,28,29,45,47,49). Multiple forms of 20a-HSDH have been distinguished in several tissues, largely on the basis of steroid substrate specificity, pyridine nucleotide requirement, and intracellular location (2, 12, 37).…”

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confidence: 99%

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens

Krafft

Hylemon

1989

J Bacteriol

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We have purified a steroid-inducible 20a-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%.Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17ai,21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 Clostridium scindens is the only bacterium isolated known to synthesize 20a-hydroxysteroid dehydrogenase (20a-HSDH) and steroid-17-20-desmolase activities (3, 51). Preliminary studies with cell extracts indicated that both neutral steroid-transforming activities were coinducible in C. scindens cultured in the presence of specific C21 steroids. In addition, it was found that both conversions required a pyridine nucleotide coenzyme, bivalent metal cations, and the same adrenocorticosteroid substrates for maximal activity (22). 20a-HSDH (EC 1.1.1.149) is widely distributed in nature. The enzyme has been found previously in bird testes, fungi (8, 11), and a great variety of mammalian tissues. In vertebrate species, 20a-HSDH is thought to be a key enzyme involved in tissue-specific regulation of steroid hormone metabolism (10, 39). Various forms of 20a-HSDH are found in the major steroid-producing tissues of mammals (adrenals, ovaries, testes, and placenta) and in liver, kidney, muscle, lymphatic organs, fibroblasts, and hematopoietic cells (4,28,29,45,47,49). Multiple forms of 20a-HSDH have been distinguished in several tissues, largely on the basis of steroid substrate specificity, pyridine nucleotide requirement, and intracellular location (2, 12, 37). A soluble enzyme from human placenta (36), rat ovary (35,50), and the testes of two species (33, 40) has been purified to homogeneity and well characterized. This report describes the purification and partial characterization of a novel form of 20a-HSDH from C. scindens.( corticosterone, 11,21-dihydroxy-4-pregnene-3,20-dione; deoxycorticosterone, 21-hydroxy-4-pregnene-3,20-dione; progesterone, 4-pregnene-3,20-dione; 17a-hydroxyprogesterone, 17a-hydroxy-4-pregnene-3,20-dione; pregnenolone, 3p-hydroxy-5-pregnen-20-one; 20a-dihydrocortisol, 11P,17,20a,21-tetrahydroxy-4-pregnene-3-one; MES, MOPS,propane-sulfonic acid); CHES, (2[N-cyclohexylamino]-ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Chemicals. The following chemicals were purchased: unlabeled steroids, Steraloids Inc., Wilton, N.H., and Sigma Chemical Co., St. Louis, Mo.; [1,2-3H]cortisol (40.6 Ci/ mmol), Amersham Corp; high-pressure liquid chromatography (HPLC) solvents, Burdick and Jackson; brain-heart infusion medium, Difco Laboratories; protease inhibitors, Boehringer Mannheim; pyridine nucleotide...

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Separation of a purified adrenal 20α-hydroxysteroid dehydrogenase

Cited by 15 publications

References 5 publications

Molecular cloning of cDNA for rat ovarian 20 α-hydroxysteroid dehydrogenase (HSD1)

Molecular cloning of cDNA for rat ovarian 20 α-hydroxysteroid dehydrogenase (HSD1)

Characterization of a human 20alpha-hydroxysteroid dehydrogenase

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens

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