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PubMed2001

Expression and putative role of 11 beta-hydroxysteroid dehydrogenase isozymes within the human eye.

PURPOSE: The human eye is an important target tissue for steroid hormones, and glucocorticoids have been implicated in the pathogenesis of ocular disease, including glaucoma. In peripheral tissues, corticosteroid hormone action is regulated at a prereceptor level through the activity of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isozymes: an oxo-reductase (11 beta-HSD1) that activates cortisol (F) from cortisone (E) and a dehydrogenase (11 beta-HSD2) that inactivates F to E. The purpose of this study was to analyze the expression and putative role of 11 beta-HSD within the human eye. METHODS: Immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed on sections of human ocular tissues, surgical trabecular meshwork (TM) specimens and a ciliary nonpigmented epithelial (NPE) cell-line. Free F and E concentrations in aqueous humor were determined by gas chromatography-mass spectrometry (GC/MS). IOP was measured in eight male volunteers before and after oral ingestion of carbenoxolone (CBX), a known inhibitor of 11 beta-HSD. RESULTS: 11 beta-HSD1 was expressed in the basal cells of the corneal epithelium and the NPE. 11 beta-HSD2 was restricted to the corneal endothelium. RT-PCR revealed mRNA for only the glucocorticoid receptor (GR) in the TM specimens, whereas GR, mineralocorticoid receptor and 11 beta-HSD1 mRNAs were all present in the NPE cell line. The demonstration of free F in excess of E (F/E 14:1) in the aqueous humor suggested predominant 11 beta-HSD1 activity. Compared with baseline (14.7 +/- 1.06 mm Hg, mean +/- SD), the IOP decreased significantly on both the third and seventh days of CBX ingestion (12.48 +/- 1.11 mm Hg, P < 0.0001 and 11.78 +/- 1.50 mm Hg, P < 0.0001, respectively). CONCLUSIONS: These results suggest that the 11 beta-HSD1 isozyme may modulate steroid-regulated sodium transport across the NPE, thereby influencing IOP.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2002

Human adrenal cortex and aldosterone secreting adenomas express both 11beta-hydroxysteroid dehydrogenase type 1 and type 2 genes.

11beta-Hydroxysteroid dehydrogenase type 1 and type 2 (11betaHSD1 and 11betaHSD2) isozymes catalize the conversion of inactive glucocorticoids (e.g. cortisone) to their active forms (e.g. cortisol) and vice versa, respectively. Reverse transcription-polymerase chain reaction allowed the detection of 11betaHSD1 and 11betaHSD2 mRNAs in the human adrenal cortex, liver, kidneys, as well as in six aldosterone-secreting adenomas. 11betaHSD1 and 11betaHSD2 activity, as evaluated by the capacity of the microsomal fraction to convert [3H]cortisone to [3H]cortisol and vice versa, was detected in both human adrenal cortex and aldosteronomas, although it was less elevated than in liver and kidneys. Aldosteronomas possessed more intense 11betaHSD1 activity and less intense 11betaHSD2 activity than the normal adrenal cortex. The hypothesis is advanced that the elevated local concentration of steroid-hormone intermediates occurring in aldosteronomas up-regulates 11betaHSD1 and down-regulates 11betaHSD2, thereby contributing to their enhanced steroidogenic function.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1992

Androgen hydroxysteroid dehydrogenases under the influence of pyridoxine derivatives.

The metabolism of testosterone in the rat ventral prostate, anterior pituitary, basal hypothalamus and amygdala was studied in vitro under the influence of vitamin B6 compounds. The influence of these compounds on the activity of 5 alpha-reductase (5 alpha-R), 3 alpha- and 17 beta-hydroxysteroid dehydrogenase (3 alpha-HSD, 17 beta-HSD) was determined for all the examined tissues. Pyridoxine hydrochloride significantly increased the activity of 5 alpha-R, 3 alpha- and 17 beta-HSD, but pyridoxal hydrochloride had an inhibitory influence on 5 alpha-R and showed no effect on 3 alpha-HSD activity at the prostate level. Male rat anterior pituitary, basal hypothalamus or amygdala incubated with pyridoxal phosphate and pyridoxal hydrochloride showed modified enzymatic activities. Pyridoxal hydrochloride showed an inhibitory effect on 5 alpha-R in the rat pituitary and basal hypothalamus as well as in the rat prostate.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

Hydroxysteroid dehydrogenases in the interrenal gland and the ovary of stork-billed kingfisher, Pelargopsis capensis (Linn.): a histochemical study.

The distribution of delta5 3beta-hydroxüsteroid dehydrogenase (delta5 3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADH-diaphorase enzymes has been histochemically studied in the interrenal gland and the ovary of the stork-billed kingfisher, Pelargopsis capensis (Linn.). In the interrenal gland, the activity for all the enzymes studied, occurred in the interrenal cells. All these enzyme activities occurred in the theca interna of normal growing follicles, atretic follicles and interstitial gland cells of the ovary. A weak activity for all the enzymes occurred in the hypertrophied granulosa cells of the atretic follicles. The significance of these findings is discussed.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Mechanism of adrenal insufficiency following trauma and severe hemorrhage: role of hepatic 11beta-hydroxysteroid dehydrogenase.

BACKGROUND: Although adrenal insufficiency may not occur with moderate hypotension, it does occur with severe hemorrhage. Since hepatocellular function is depressed following severe hemorrhage, it remains unknown whether the liver plays any role in regulating adrenal function after trauma and hemorrhagic shock. HYPOTHESIS: Hepatic 11beta-hydroxysteroid dehydrogenase (11beta-HSD), a microsomal enzyme responsible for the degradation of bioactive corticosterone, plays a major role in the development of adrenal insufficiency following trauma and severe hemorrhage. DESIGN, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Male rats underwent laparotomy to induce trauma before hemorrhage. They were then bled to and maintained at a blood pressure of 40 mm Hg until 40% of the maximal bleed-out volume was returned in the form of Ringer lactate. The rats were then resuscitated with 4 times the volume of maximal bleed-out with Ringer lactate during a 60-minute period. Plasma levels of corticosterone and corticotropin were measured at various intervals. In additional groups, corticotropin-induced corticosterone release, adrenal contents of corticosterone and cyclic adenosine monophosphate (cAMP), hepatic 11beta-HSD activity, and plasma levels of corticosterone-binding globulin were determined at 1.5 hours after resuscitation. Moreover, a model of moderate hypotension (blood pressure, 80 mm Hg) was used to determine whether adrenal function is depressed under such conditions. RESULTS: At the time of maximal bleed-out, plasma corticosterone and corticotropin levels increased by 245% (P<.001) and 293% (P<.001), respectively. Despite corticotropin levels being similar to those of the animals undergoing sham operation after resuscitation, corticosterone levels in hemorrhaged animals remained elevated up to 4 hours after resuscitation (by 158%-207%; P<.001). In addition, corticotropin-induced corticosterone release decreased by 78% at 1.5 hours after resuscitation (P = .009). In contrast, moderate hypotension did not reduce corticotropin-induced corticosterone release. Adrenal corticosterone content and cAMP levels (i.e., the second messenger of corticotropin action) decreased by 55% (P<.001) and 25% (P = .03), respectively. Hepatic 11beta-HSD activity decreased significantly at 1.5 hours after resuscitation (P<.001). CONCLUSIONS: Sustained increase in plasma corticosterone levels following hemorrhage and resuscitation may be, in part, due to the decreased hepatic 11beta-HSD activity. The high level of corticosterone negatively regulates corticotropin release, further reducing adrenal responsiveness to corticotropin stimulation. Thus, the liver appears to play an important role in regulating adrenal function following trauma and severe hemorrhage.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Expression of 11 beta-hydroxysteroid dehydrogenase in rat osteoblastic cells: pre-receptor regulation of glucocorticoid responses in bone.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) acts as a pre-receptor signaling mechanism for corticosteroids by regulating the access of active glucocorticoids to both glucocorticoid (GR) and mineralocorticoid receptors (MR). To examine the relationship between endogenous glucocorticoid metabolism and osteoblast function, we have characterized the expression of 11 beta-HSD isozymes in rat osteosarcoma cells. Analysis of mRNA from ROS 25/1, UMR 106 and ROS 17/2.8 cells revealed transcripts for both 11 beta-HSD type 1 (11 beta-HSD1) and type 2 (11 beta-HSD2) in all three cell lines. However, enzyme activity studies showed only high affinity dehydrogenase activity (inactivation of corticosterone (B) to 11-dehydrocorticosterone (A)), characteristic of 11 beta-HSD2; conversion of B to A was higher in ROS 25/1> UMR 106 cells>ROS 17/2.8. Although all three cell lines had similar numbers of GR (50,000/cell), glucocorticoid modulation of alkaline phosphatase activity and cell proliferation was only detectable in ROS 17/2.8 cells. Further studies showed that 11 beta-HSD2 activity in each of the cells was potently stimulated by both A and B, but not by synthetic dexamethasone. This effect was blocked by the 11 beta-HSD inhibitor, 18 beta-glycyrrhetinic acid (but not by GR or MR antagonists) suggesting direct, allosteric regulation of 11 beta-HSD2 activity. These data indicate that in osteosarcoma cells 11 beta-HSD2 plays a key role in controlling GR-mediated responses; cells with relatively high levels of 11 beta-HSD2 activity were insensitive to glucocorticoids, whilst cells with low levels showed functional responses to both dexamethasone and B. In addition to the established effects of 11 beta-HSD2 in protecting MR in the kidney and colon, our data suggest that 11 beta-HSD2 in bone represents an important pre-receptor mechanism in determining ligand availability to GR.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1989

Determination of bile acids and their conjugates in serum by HPLC using immobilized 3 alpha-hydroxysteroid dehydrogenase for detection.

A method is described for the separation of unconjugated bile acids and their glycine and taurine conjugates in a single step with adequate sensitivity for analysis of serum samples. Separation was carried out over a period of 80 min using a linear gradient with increasing concentrations of methanol in aqueous ammonium dihydrogen phosphate buffer, on an ODS Spherisorb column. Spectrofluorometric detection of NADH, formed as the column eluate passed through a column of immobilized 3 alpha-hydroxysteroid dehydrogenase, enabled amounts less than 40 pmol to be quantified. The reaction was carried out at neutral pH so that the lifetime of the enzyme column was increased, compared to other methods where a pH nearer 9.0 is commonly used. Flow rates were optimized to give comparable peak area for both primary and secondary bile acids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Inhibition of 11beta-hydroxysteroid dehydrogenase by bile acids in rats with cirrhosis.

Renal sodium retention and potassium loss occur early, in many instances in the preascitic state of cirrhosis, an observation that cannot be fully explained by increased aldosterone concentrations. We therefore hypothesize that 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2), which protects mineralocorticoid receptors (MR) from glucocorticosteroids, is down-regulated in cirrhosis. Cirrhosis was induced by bile duct ligation in rats. The urinary ratio of (tetrahydrocorticosterone + 5alpha-tetrahydrocorticosterone)/ 11-dehydro-tetrahydrocorticosterone [(THB+5alpha-THB)/THA] was measured by gas chromatography. Cortical collecting tubules (CCT) were isolated by microdissection and used for measurements of the activity of 11beta-HSD2 by assessing the conversion of corticosterone to dehydrocorticosterone. The mRNA content of 11beta-HSD2 was determined by reverse-transcription polymerase chain reaction (RT-PCR) in CCTs. The urinary ratio of (THB+5alpha-THB)/THA increased concomitantly with the urinary excretion of bile acids following bile duct ligation. Chenodeoxycholic acid (CDCA) dose-dependently inhibited 11beta-HSD2 in CCT with a Ki of 19.9 micromol/L. Four weeks after bile duct ligation, 11beta-HSD2 activity was decreased in CCT, an observation preceded by a reduced mRNA content at weeks 2 and 3. In cirrhosis, the MR-protecting effect by 11beta-HSD2 is diminished, and therefore, endogenous glucocorticoids can induce MR-mediated sodium retention and potassium loss.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Protein kinase C increases 11beta-hydroxysteroid dehydrogenase oxidation and inhibits reduction in rat Leydig cells.

Glucocorticoid hormone controls Leydig cell steroidogenic function through a receptor-mediated mechanism. The enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) plays an important role in Leydig cells by metabolizing glucocorticoids, and catalyzing the interconversion of corticosterone (the active form in rodents) and 11-dehydrocorticosterone (the biologically inert form). The net direction of this interconversion determines the amount of biologically active ligand, corticosterone, available for glucocorticoid receptor binding. We hypothesize that 11betaHSD oxidative and reductive activities are controlled separately in Leydig cells, and that shifts in the favored direction of 11betaHSD catalysis provide a mechanism for the control of intracellular corticosterone levels. Therefore, in the present study, we tested the dependency of 11betaHSD oxidative and reductive activities on protein kinase C (PKC) and calcium-dependent signaling pathways. 11betaHSD oxidative and reductive activities were measured in freshly isolated intact rat Leydig cells using 25 nM radiolabeled substrates after treatment with protein kinase modulators. We found that PKC and calcium-dependent signaling had opposing effects on 11betaHSD oxidative and reductive activities. Stimulation of PKC using the PKC activator, 6-[N-decylamino]-4-hydroxymethylinole (DHI), increased 11betaHSD oxidative activity from a conversion rate of 5.08% to 48.23% with an EC50 of 1.70 +/- 0.44 microM (mean +/- SEM), and inhibited reductive activity from 26.90% to 3.66% conversion with an IC50 of 0.22 +/- 0.05 microM. This indicated that PKC activation in Leydig cells favors 11betaHSD oxidation and lower levels of corticosterone. The action of DHI was abolished by the PKC inhibitor bisindolylmaleimide I. In contrast, addition of calcium to Leydig cells increased 11betaHSD reductive activity while decreasing oxidative activity, thereby favoring reduction and conversion of inert 11-dehydrocorticosterone into active corticosterone. The opposite effect was seen after elimination of calcium-dependent signaling, including removal of calcium by EGTA or addition of the calmodulin (calcium binding protein) inhibitor SKF7171A, or the calcium/calmodulin-dependent protein kinase I (CaMK II) inhibitor, KN62. We conclude that 11betaHSD oxidative and reductive activities are separately regulated and that, in contrast to calcium-dependent signaling, PKC stimulates 11betaHSD oxidation while inhibiting 11betaHSD reduction. Maintenance of a predominantly oxidative 11betaHSD could serve to eliminate adverse glucocorticoid-induced action in Leydig cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2002

ATP stimulates human placental 11beta-hydroxysteroid dehydrogenase type 2 activity by a novel mechanism independent of phosphorylation.

The human placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is believed to play a key role in fetal development since this enzyme protects the fetus from exposure to high levels of maternal cortisol by virtue of converting maternal cortisol to its inert metabolite cortisone. The present study was undertaken to examine the effect of ATP on 11beta-HSD2 activity in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was stimulated more than six-fold by 0.5 mM ATP (EC(50) = 0.2 mM). Such stimulation appears to be mediated through a novel mechanism independent of ATP-induced phosphorylation of the reaction components since AMP-PNP, a non-hydrolyzable analogue of ATP, was equally effective. The ATP-induced stimulation of 11beta-HSD2 activity is adenine nucleotide specific in that a similar stimulation was observed with ADP and AMP but not with CTP, GTP, or UTP. Furthermore, ATP increased the maximal velocity (V(max)) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the apparent K(m) of 11beta-HSD2 for cortisol, suggesting that ATP may stimulate enzyme activity by interacting with the enzyme at a site other than that involved in substrate binding. In conclusion, the present study has identified ATP as a novel regulator of human placental 11beta-HSD2 in vitro. It is conceivable that intracellular ATP may have a profound effect on 11beta-HSD2 function in vivo.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2002

Postnatal development of 11beta-hydroxysteroid dehydrogenase type 1 in the rat hippocampus.

Glucocorticoids (GCs) have important actions in the hippocampus of the brain, where their access to glucocorticoid receptor (GR) is increased by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 11beta-HSD1 converts biologically inactive 11-dehydrocorticosterone into active corticosterone. However, the postnatal development of 11beta-HSD1 in the hippocampus is not properly understood. In this study, the postnatal distribution and development of 11beta-HSD1 in the hippocampus of the rat brain was studied with immunohistochemistry and Western blot analysis. Results showed that abundant 11beta-HSD1 immunoreactive substance (ir-11beta-HSD1) was present in the hippocampus. There were homogeneous distributions of 11beta-HSD1 in the hippocampal CA1, CA2, CA3, CA4 regions and the dentate gyrus at postnatal days 1, 3, and 7. Interestingly, the developmental distribution of GR in the hippocampus followed the same pattern as 11beta-HSD1. Western blot analysis demonstrated that a higher level of expression of 11beta-HSD1 in the hippocampus was found in the first 2 weeks of life. The expressions of 11beta-HSD1 started to drop to adult levels at about postnatal day 15 both in the hippocampus and in other brain areas. These results suggest that the higher expression of 11beta-HSD1 in the neonatal hippocampus may be important for the maturation of the central nervous system mediated by GCs through GR.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1992

Inhibition of 3 alpha-hydroxysteroid oxidoreductase and 5 alpha-reductase activity by antiandrogens and indomethacin in the rat prostate.

We studied the effects of antiandrogens in vitro on inhibition of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) and 5 alpha-reductase activities in the rat prostate. Kinetic and inhibition experiments were analyzed by thin layer chromatography. Cyproterone acetate (CA), chlormadinone acetate (CMA), and TZP-4238 (TZP), which is more potent than other antiandrogens, were used as inhibitors and were compared with indomethacin IND, which is a recognized 3 alpha-HSOR inhibitor. The IC50S of CA, CMA, IND, and TZP for 3 alpha-HSOR reductase in cytosol were about 5, 10, 10, and 100 microM, respectively, and inhibition was competitive. The IC50 of IND for 3 alpha-HSOR reductase in microsomes was 20 microM. The IC50S of other inhibitors were > 100 microM, and inhibition was noncompetitive. The IC50S of CA, CMA, IND, and TZP for 3 alpha-HSOR oxidase in cytosol were > 100 microM, and inhibition was competitive or noncompetitive. Inhibition of 3 alpha-HSOR oxidation was not observed in microsomes. The difference between these inhibition patterns suggests that there may be 4 isoenzymes in rat prostatic tissue. The IC50S of MK-906, CMA, and TZP for 5 alpha-reductase in prostate homogenate were about 0.01, 200, and 200 microM, respectively.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Presence of 11 beta-hydroxysteroid dehydrogenase enzyme in the human prostate tumor cell line LNCaP.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is the enzyme that catalyzes the reversible oxidation of the biologically active steroid cortisol and corticosterone to their inactive metabolites cortisone and dehydrocorticosterone. We report its presence in significant levels in the human prostate carcinoma cell line LNCaP cultured in medium RPMI-1640 with 10% fetal calf serum (FCS). The 11-dehydrogenase activity of 11 beta-HSD is present, while the 11-reductase activity is undetectable in these tumor cells under the present culture conditions. The enzyme activity is found to be linear with time of incubation, and is proportional to plated cell density. The enzymatic activity can be determined in cultures maintained for longer times. Carbenoxolone, the potential inhibitor of the 11 beta-HSD, inhibits 95% of the dehydrogenase activity of the tumor cells when used in nM concentration. The presence of this enzyme in tumor cell line indicates that 11 beta-HSD plays an important role in maintaining the active glucocorticoid levels in the prostate.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Prolactin-mediated inhibition of 20alpha-hydroxysteroid dehydrogenase gene expression and the tyrosine kinase system.

The rat luteal 20alpha-hydroxysteroid dehydrogenase plays a key role at catabolizing progesterone and at decreasing the level of this steroid secreted by the ovaries. Throughout pregnancy and before parturition neither the mRNA nor the protein for this enzyme could be detected. In this investigation we set to examine whether PRL and PRL-like hormone from placental origin silence the expression of this gene and whether PRL action involves tyrosine kinase activity and/or de novo protein synthesis. The results revealed that PRL and PRL-like hormone from rat placental origin (rPL-1 and rPL-2), but not rat growth hormone, caused a rapid and profound inhibition of 20alpha-HSD mRNA expression in highly luteinized granulosa cells. Immunoprecipition and western blot analysis indicate that PRL-R associates with JAK2 and Stat5, and this association is increased within 30 seconds with PRL treatment. Although both JAK2 and Stat5 were phosphorylated on tyrosine upon PRL treatment, the PRL mediated inhibition of 20alpha-HSD was not reversed by either tyrosine kinase inhibitors, AG18 and genistein, but was largely reversed by the protein synthesis inhibitor cycloheximide. In summary, results of this investigation indicate that although PRL can activate the JAK2/Stat5 system in the corpus luteum, the down regulation of 20alpha-HSD mRNA by PRL does not appear to involve tyrosine kinase activity but depends on de novo synthesis of protein(s).

20-Hydroxysteroid Dehydrogenases↗
PubMed1998

Molecular cloning and characterization of the rat ovarian 20 alpha-hydroxysteroid dehydrogenase gene.

The rat 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is an enzyme responsible for the catabolism of progesterone to the inactive 20 alpha-hydroxprogesterone. We have previously shown that the expression of this enzyme is not regulated by post-translational modification, but at the level of transcription. In this study we have established that the 20 alpha-HSD gene contains nine exons and have isolated a 2.5 kb promoter region. The transcription start site was identified and a TATA box was found. 5' deletions of this promoter significantly decreased basal promoter activity. Treatment with forskolin led to a dose dependent inhibition of the 2.5kg-20 alpha-HSD-luciferase construct. Computer analysis identified one CRE, two Nur77 response elements, two putative AP1 sites and one progesterone response element half-site. In summary, we have identified and partially characterized the promoter region of the rat ovarian 20 alpha-HSD and demonstrated that the regulatory elements for 20 alpha-HSD are present within a 2.5 kb 5' flanking region of the gene.

20-Hydroxysteroid Dehydrogenases↗
PubMed1998

Serines at the active site of 11 beta-hydroxysteroid dehydrogenase type I determine the rate of catalysis.

Short chain alcohol dehydrogenases have an invariant YXXXK motif at the active site. Database analysis of 116 superfamily members showed that 92% also contain a Serine or Threonine residue at the Y + 1 or Y + 3 positions, a pattern we previously described as the ST rule. In the present study we have mutated Serines in the active site, YSASK, motif of 11 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studies were facilitated by the generation of a new specific polyclonal antibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34 kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active site Serines resulted in modest increases of Km values for corticosterone from 325 nM for 11 beta HSD1 to 512 nM-588 nM for the S1 (YAASK), S2 (YSAGK) and S3 (YAAGK) mutants in homogenates of transfected CHOP cells. However, far greater effects were observed on the first order rate constants with mutants displaying 10%, 1% and 1% of the wild type activity, respectively. When the oxidoreductase reaction was studied in whole cells mutagenesis again had a minimal effect on the Km value but dramatically lowered first order rate constants to 34%, 5% and 6%, respectively, of the wild type. These data show that Serines at the active site of 11 beta HSD1 play an important role in determining the rate of catalysis. Coexpression of wild type and mutant enzymes did not lower wild type activity suggesting that the active site of the multimeric enzyme is not a composite of active site Serines from different subunits.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Expression of human kidney 11beta-hydroxysteroid dehydrogenase (11-HSD2) in bacteria.

The kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) protects the mineralocorticoid receptor from spurious activation by glucocorticoids. To explore structure-function relationships, human 11-HSD2 cDNA was subcloned into the bacterial expression vector, pET25b. E. coli transformed with wild-type cDNA produced active enzyme that retained biochemical characteristics of the native protein. The addition of 6 histidine residues to the C-terminus of the wild-type enzyme (11-HSD2/His) increased activity 2-fold. Whereas wild-type activity was almost completely sedimented following 100,000g centrifugation, 10-30% of total activity of 11-HSD2/His remained in the supernatant. The 11-HSD2 isozyme normally contains three N-terminal hydrophobic domains. Mutant 11-HSD2/His possessing a single hydrophobic domain retained partial activity, but elimination of all domains inactivated the enzyme. Thus, the N-terminal hydrophobic domains are essential for complete activity of 11-HSD2 but association with an intact cell membrane is not.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗