Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the interconversion of cortisol to hormonally inactive cortisone (corticosterone (B) to 11-dehydrocorticosterone (A) in rodents), and as such is established as a pre-receptor signalling pathway for corticosteroid hormone action. To further evaluate the role of this enzyme in adult and fetal life we have characterized two isoforms of 11beta-HSD in mouse tissues. Mouse 'liver' or type 1 11beta-HSD is a bi-directional dehydrogenase/oxo-reductase (K(m) for B 1.9 microM, K(m) for A 0.73 microM). Oxo-reductase activity utilized only NADPH as a co-factor, whilst dehydrogenase activity increased with both NAD or NADP. Mouse 'kidney' or 11beta-HS3D2 activity was NAD-dependent with a K(m) for B of 0.11 microM. Dexamethasone was not a substrate. Using an in-house mouse 11beta-HSD2 cDNA and NAD-dependent activity studies, 11 beta-HSD2 was expressed in epithelial cells of colon, renal collecting ducts, ovary, and adrenal, but was absent in liver, spleen, testis and heart. With the exception of gonadal tissues, activity and mRNA levels were consistently higher in adult male versus female tissues. In fetal kidney and colon there was absent/low levels of 11beta-HSD2 expression from fetal day 15 to term (day 19/20). Placental 11beta-HSD2 mRNA and activity were highest on fetal day 13/14 and fell progressively to undetectable levels by term. Two isoforms of 11beta-HSD are present in mouse tissues in accordance with other mammalian species. The sexual-dimorphic expression 11 beta-HSD2 in kidney and colon may reflect male-female differences in sodium homeostasis, and the absent expression of 11 beta-HSD2 in late gestation may facilitate glucocorticoid-dependent maturation of mouse fetal tissues.
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PMID:Ontogeny and sexual dimorphic expression of mouse type 2 11beta-hydroxysteroid dehydrogenase. 909 7

Endogenous glucocorticoids are converted to their biologically inert 11-dehydroderivatives by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). The low-K(m), NAD(+)-dependent renal isoform (Type 2) identified in the distal nephron protects mineralocorticoid receptors from activation by endogenous glucocorticoids. The function of high-K(m), NADP(+)-dependent renal isoform (Type 1) is less well understood. Since glucocorticoids may modulate sodium transport in renal proximal tubules (PT), we hypothesized that Type 1 activity in this segment may be regulated by dietary Na(+)-11 beta-HSD activity was assessed in homogenates of canine PT by the conversion of cortisol to cortisone in the presence of NADP+ 200 microM. A high-Na+ diet for 4 days increased the Vmax 4-fold, with no change in the Type 1 K(m) (40 mEq/day Na+ diet: K(m) 0.959 microM, Vmax 3.40 pmoles/min/mg protein versus 150 mEq/day Na+ diet: K(m) 0.962 microM, Vmax 14.8 pmoles/min/mg protein). Type 1 mRNA also rose in the salt repleted animals. The high-Na+ diet produced no detectable change in the Type 2 isoform enzyme kinetics and mRNA level. No reverse oxo-reductase activity was noted with either renal isoform. Thus, renal Type 1 11 beta-HSD can be regulated by dietary Na+ independent of changes in the renal Type 2 isoform.
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PMID:Influence of dietary sodium on the renal isoforms of 11 beta-hydroxysteroid dehydrogenase. 911 24

Recently, two distinct isoenzymes of 11beta-hydroxysteroid-dehydrogenase (11beta-HSD) have been cloned and characterized in several species: The isoenzyme 11beta-HSD-I is widely distributed, bidirectional, prefers NADP(H) and has a low substrate affinity. The isoenzyme 11beta-HSD-II seems to exclusively oxidize physiological glucocorticoids, uses NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroids fluorinated in position 9, however, are rapidly reduced by human kidney cortex slices. We attempted to find out which isoenzyme is responsible for this unexpected reductase activity. We studied the 11beta-HSD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidney cortex. For the reaction E to F (not for DH-D to D!), glucose-6-phosphate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/NADPH-regenerating system. Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael's constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmax(oxidation)/Vmax(reduction) (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmax(oxidation)/Vmax(reduction) was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner. In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11beta-HSD-II. The NADH-dependent 11beta-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11beta-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11beta-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.
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PMID:Metabolism of dexamethasone in the human kidney: nicotinamide adenine dinucleotide-dependent 11beta-reduction. 914 56

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11beta-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11beta-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11beta-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11beta-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11beta-HSD and MR messenger RNAs (mRNAs), and for type I 11beta-HSD protein. Leydig cell 11beta-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11beta-HSD is present in rat Leydig cells. However, unlike the type I 11beta-HSD in liver parenchymal cells, the Leydig cell 11beta-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 +/- 9.3 nM and maximum velocity (Vmax) of 7.1 +/- 1.2 pmol x min x 10(6) cells. The second component consisted of high Km activities that were consistent with type I:NADP-dependent oxidative activity with Km of 5.87 +/- 0.46 microM and Vmax of 419 +/- 17 pmol x min x 10(6) cells, and NADPH-dependent reductive activity with Km of 0.892 +/- 0.051 microM and Vmax of 117 +/- 6 pmol x min x 10(6) cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11beta-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11beta-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.
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PMID:Identification of a kinetically distinct activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells. 916 33

In the human and in rodents like the rat and mouse, the liver enzyme 11 beta-hydroxysteroid dehydrogenase type I (11 beta-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11 beta-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular, mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11 beta-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10(-6) mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10(-3) mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with K(m) values of 0.64 mumol/l and 9.8 mumol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate K(m) was 7.2 mumol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar K(m) and Vmax values for NAD+ to NADP+ as cosubstrates (K(m) of 4.3 mumol/l and 5.0 mumol/l respectively). The NAD+ preference for the oxidation of 10(-6) mol/l cortisol described above may be due to a second, NAD(+)-preferring 11 beta-HSD with a K(m) of 1.4 mumol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (K(m): 1.2 mumol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11 beta-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD(+)-preferring. This kidney enzyme (analogous to 11 beta-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.
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PMID:Evidence for isoforms of 11 beta-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig. 916 19

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described and cloned. The liver-type, NADP(+)-dependent 11 beta-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD(+)-dependent 11 beta-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11 beta-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the "gatekeeper" for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD(+)-dependent 11 beta-HSD-2 has been reported in the sheep kidney. We found both 11 beta-HSD NAD(+)- and NADP(+)-dependent activities in sheep kidney to be present. The NAD(+)-dependent activity exhibited a Km similar to that reported in the literature, 3.85 +/- 1.28 nM for corticosterone and 21.3 +/- 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP(+)-dependent activity had a Km for corticosterone of 4 +/- 1.3 nM for a Km for cortisol of 35.2 +/- 2 nM, 100-fold lower than that described for the 11 beta-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD(+)-dependent activity was approximately 3-10 times greater than the NADP(+)-dependent activity when incubated with 5 nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11 beta-HSD-2 plasmid exhibited a marked preference for NAD+ as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP+ was present, but minimal; NADP+ did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP(+)-dependent enzyme, 11 beta-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.
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PMID:The sheep kidney contains a novel unidirectional, high affinity NADP(+)-dependent 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD-3). 917 32

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.
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PMID:Intracellular regulation of 17 beta-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells. 920

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn394-Leu395-Ser396) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.
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PMID:N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2. 929 Nov 87

Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) which converts active glucocorticoids to inactive metabolites. As 11 beta HSD is also expressed in the intestine, this enzyme may be an important regulator of intestinal maturation. To investigate this, we have performed the systematic study of the development of intestinal 11 beta HSD activity and its cofactor preference as well as of the effect of 11 beta HSD inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and Na,K-ATPase in the intestine. The activity of 11 beta HSD was low in ileum of suckling rats and significantly increased during the weaning period. In colon, the activity was already high in suckling rats and gradually rose during the postnatal development. 11 beta HSD activity was undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic 11 beta HSD utilized predominantly NAD as a cofactor, but displayed significant sensitivity also to NADP. Ileal 11 beta HSD had similar sensitivity to both cofactors. With NAD as a cofactor, ileal 11 beta HSD had a Km (59 +/- 10 nM) compatible with the colonic enzyme (81 +/- 14 nM). Carbenoxolone administration to suckling and weanling rats in vivo did not result in any changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone significantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-affinity type of 11 beta HSD is expressed not only in colon but also in ileum and that 11 beta HSD is an important factor in the regulation of tissue levels of active glucocorticoids in developing colon but not in the small intestine.
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PMID:The role of 11 beta-hydroxysteroid dehydrogenase in maturation of the intestine. 937 10

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11beta-HSD is present, which, in contrast to the type 1 11beta-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11beta-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11Beta-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11beta-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11beta-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11beta-HSD2 using a chimera encoding 11beta-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11beta-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11Beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11beta-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11beta-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11beta-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11beta-HSD activity.
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PMID:The role of 11beta-hydroxysteroid dehydrogenase in steroid hormone specificity. 969 85


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