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PubMed1975

Studies on the human testis. VI. NADH-linked reactions of microsomal steroid 20alpha-and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase.

NADH-linked 20alpha- and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase activities were demonstrated in the microsomal fraction of the human testis. The microsomal 20alpha-hydroxysteroid dehydrogenase showed substrate affinity to pregnenolone and progesterone and not to 17alpha-hydroxyprogesterone and preferred NADH to NADPH as a hydrogen donor. In the presence of NADH, the optimal pH for the enzyme was 7.7 and the apparent Michaelis constants of the enzyme for progesterone and pregnenolone at 37 C and pH 7.4 were 6.9-7.1 X 10-6M and in the order of 10-5M, respectively, 17alpha, 20beta-Dihydroxypregn-4-en-3-one was the only significant metabolite produced from 17alpha-hydroxyprogesterone by microsomal fraction of the human testis in the presence of NADH. The apparent Michaelis constant of microsomal 20beta-hydroxysteroid dehydrogenase for 17alpha-hydroxyprogesterone in the presence of NADH was in the order of 10-5M at 37 C and pH 7.4. The microsomal 17alpha-hydroxylase catalyzed the metabolism of pregnenolone and progesterone at a similar rate in the presence of NADH. The optimal pH and the apparent Michaelis constant at 37 C and pH 7.4 of the NADH-linked reaction of 17alpha-hydroxylase for progesterone were 7.7 and 5.3-5.4 X 10-7M, resepctively. The NADH-linked enzyme activity for progesterone was competitively inhibited by both pregn-5-ene-3beta, 20alpha-diol (inhibition constant: 1.7 X 10-7M) and 20alpha-hydroxypregn-4-en-3 one (inhibition constant: 6.6 X 10-7M), and was resistant to poor oxygen supply during incubation. The results indicate that the microsomal 20alpha-hydroxysteroid dehydrogenase is a different enzyme from the one in the soluble fraction of the human testis and that microsomal 17alpha-hydroxylase in the human testis is activated by NADH as well as NADPH.

Binding, Competitive↗

PubMed1984

Relationship between glucose and 20 alpha-hydroxysteroid dehydrogenase in fetal sheep erythrocytes.

We have examined whether glucose supply to fetal sheep erythrocytes limits the rate of 20 alpha-reduction of progesterone in blood and as such is associated with the progressive loss of 20 alpha-hydroxysteroid dehydrogenase activity which has been observed from 30 days before term. Enzyme activity in erythrocytes depleted of glucose by washing was regained in the presence of at least 0.167 mmol glucose/1. The cofactor NADPH was necessary to support the reaction in lysed cells. Addition of glucose to whole blood diluted 20-fold for assay of 20 alpha-hydroxysteroid dehydrogenase did not increase the rate of reaction. Infusion of dextrose to increase fetal plasma glucose concentrations had no effect on 20 alpha-hydroxysteroid dehydrogenase activity. Over the period from 114 to 137 days of gestation, both dextrose- and saline-infused fetuses showed a decline in enzyme activity from a combined mean of 1.45 +/- 0.21 (S.E.M.) to a mean of 0.78 +/- 0.18 mumol/ml erythrocytes per h. Fetal leucocytes did not contribute significantly to the activity of 20 alpha-hydroxysteroid dehydrogenase in whole blood. The rate of 20 alpha-reduction of progesterone in the blood of eight fetuses with indwelling carotid catheters declined from 2.31 +/- 0.09 mumol/ml erythrocytes per h at 90-95 days of gestation to 0.73 +/- 0.04 mumol/ml per h at 141-145 days. However, a consistent decline was only observed after 116-120 days. The apparent equilibrium position for progesterone reduction to 20 alpha-dihydroprogesterone varied between 83.9 +/- 1.8 and 65.7 +/- 4.2%.(ABSTRACT TRUNCATED AT 250 WORDS)

20-Hydroxysteroid Dehydrogenases↗

PubMed1980

Enzymic color development of urinary 3 alpha-hydroxysteroids on thin-layer chromatograms.

I describe the use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) for enzymic color development of 3 alpha-hydroxysteroids. Urinary 3 alpha-hydroxysteroids are converted to 3-ketosteroids by the enzyme, and the NADH formed is reacted with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-tetrazolium chloride and "diaphorase" to form the colored formazan. The dye so formed on a thin layer chromatographic plate is measured by densitometoric scanning. I illustrate use of this method by showing results for urinary excretion of 3 alpha-hydroxysteroids by some various sorts of patients.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Variations in human placental 11 beta-dehydrogenase and 11-oxoreductase activities of 11 beta-hydroxysteroid dehydrogenase enzyme during pregnancy.

Human placental 11 beta-hydroxysteroid dehydrogenase enzyme has an important role in controlling glucocorticoids reaching the fetus. Excess glucocorticoids impair fetal growth. Recent investigations show that the placenta is rich in NAD- and NADP-dependent 11 beta-hydroxysteroid dehydrogenase activity. Elucidation of the activities of both these isoforms is necessary to understand placental glucocorticoid metabolism. Hence we determined both NAD- and NADP-dependent 11 beta-hydroxysteroid dehydrogenase activities throughout pregnancy. 11 beta-dehydrogenase (oxidative) and 11-oxoreductase (reductive) activities of 11 beta-hydroxysteroid dehydrogenase were determined in 16 first-trimester (9-12 weeks) and 14 second-trimester (13-22 weeks) and 17 term (38-42 weeks) placentae. Both NAD- and NADP-dependent activities increased with pregnancy. The second-trimester NAD-dependent activity was higher than the first-trimester activity (p = 0.02). At term this activity was higher than during the second (p = 0.05) and first (p = 0.0002) trimesters. A similar increase was obtained with NADP isoform except that the difference between first and second trimesters was not significantly different at p = 0.05. The NADH-dependent 11-oxoreductase activity was also detected throughout the pregnancy. However, the activity at term was significantly higher than during the second (p = 0.005) and first (p = 0.001) trimesters. This increase may result in a concomitant increase of cortisol reaching fetus, thus helping fetal lung maturation.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1983

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase.

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.

Animals↗

PubMed1990

Characterization of NADP+: 3 beta-hydroxysteroid dehydrogenase from microsomes of rat liver.

A NADP(+)-dependent 3 beta-hydroxysteroid dehydrogenase activity was localized in the microsomal fraction of rat liver. This enzyme was solubilized and separated completely from 3 alpha-hydroxysteroid dehydrogenase by Matrex red A column chromatography. Partially purified 3 beta-hydroxysteroid dehydrogenase catalyzed the oxidation and reduction between the 3 beta-hydroxyl and 3-ketonic group of steroids or bile acids having no double bond in the A/B ring, but was inactive toward 3 alpha-hydroxyl group. The enzyme required NADP+ for oxidation and NADPH for reduction. The activity was inhibited by p-chloromercuribenzoic acid or p-chloromercuribenzenesulfonic acid at the concentration of 10(-4) M. The molecular weight of the enzyme was estimated to be about 43,000 by Sephadex G-200 column chromatography. From these results, it is concluded that the enzyme is a new type of microsomal NADP+:3 beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1988

Mouse liver dihydrodiol dehydrogenases. Identity of the predominant and a minor form with 17 beta-hydroxysteroid dehydrogenase and aldehyde reductase.

A major and a minor form of dihydrodiol dehydrogenase were co-purified with 17 beta-hydroxysteroid dehydrogenase and aldehyde reductase, respectively, to apparent homogeneity from liver cytosol of male ddY mice. The activities of dihydrodiol dehydrogenase and testosterone dehydrogenase or aldehyde reductase of the two enzyme forms comigrated electrophoretically. The major form of the enzyme oxidized 17 beta-hydroxysteroids and nonsteroidal alicyclic alcohols and reduced 17-ketosteroids and various synthetic carbonyl compounds, showing higher affinity for steroids than for xenobiotics. The activity of this enzyme form toward benzene dihydrodiol and testosterone exhibited identical thermostability and susceptibility to inhibition by quercitrin, SH-reagents, nonsteroidal estrogens and anti-inflammatory agents. On the other hand, the minor form of the enzyme, which oxidized benzene dihydrodiol but not 17 beta-hydroxysteroids, also reduced various aldehydes well and was specifically inhibited by barbiturates and sorbinil. These results indicate that the major form of dihydrodiol dehydrogenase is identical to 17 beta-hydroxysteroid dehydrogenase and the minor enzyme form to aldehyde reductase.

Alcohol Dehydrogenase↗

PubMed1983

Microsomal 5-ane-3 beta-hydroxysteroid oxidoreductase from pubertal rat Leydig cells: partial purification and characterization.

A 3 beta-hydroxysteroid oxidoreductase which acts on 5 alpha (beta)-reduced C19 and C21 steroids (5-ane-3 beta-hydroxysteroid oxidoreductase; 5-ane-3 beta-HSO) has been solubilized from pubertal rat Leydig cell microsomes and purified 300-fold by ion exchange and gel filtration chromatography. The partially purified enzyme is stable only in the presence of 0.4 M NaCl and appears to exist as a molecule having a molecular weight of 35,000 or as aggregates with a molecular weight in excess of 150,000. NAD+ and NADH+ are used exclusively as cofactors. The velocity of the steroid oxidation reaction was unaffected by either Ca2+ or Mg2+. The steroid oxidation reaction has a pH optimum between 8.0 and 8.5, a temperature optimum at 35 degrees C and an activation energy of 12,850 cal/mol. The pH optimum of the steroid reduction reaction is 6.6. A variety of 5 alpha-reduced C19 and C21 steroids can be utilized as substrates. Treatment of microsomes with phospholipase A2 resulted in a 26 to 90% loss of enzyme activity, paralleling decreased microsomal phospholipid content, and suggesting a role for phospholipids in 5-ane-3 beta-HSO activity. Assays with combined substrates indicate that one enzyme is responsible for activities observed with 5 alpha- and 5 beta-reduced C19- and 5 alpha-reduced C21-3 beta-hydroxysteroids. Purification data indicate that the 5-ane-3 beta-HSO and the 5-ene-3 beta-hydroxysteroid oxidoreductase:isomerase are distinct enzymes.

Animals↗

PubMed1989

Expression of hydroxysteroid sulphotransferase is related to estrogen receptor status in human mammary cancer.

A positive correlation between the expression of estrogen sulphotransferase (EC 2.8: 2.4) and the estrogen receptor (ER) in human breast cancer tissues was previously demonstrated. We have now established that a similar correlation exists between the expression of hydroxysteroid sulphotransferase (EC 2.8: 2.2) and ER in such tissues. Enzyme activity was present in 93% of the ER + tumor cytosols (mean 59 +/- 44 (SD) pmol dehydroepiandrosterone sulphate formed per mg protein per 2 h (n = 42). Activity was detected in 68% of ER - tumors and this was significantly lower (mean 21 +/- 26 (SD) (n = 19), P less than 0.001) than the former group. Metabolism of estradiol-17 beta (E2) and the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL), which is a substrate for hydroxysteroid sulphotransferase but not estrogen sulphotransferase, was studied in four ER + human mammary cancer cell lines (MCF-7, T47-D, MDA-MB-361 and ZR-75-1) and four ER-human mammary cell lines (BT-20, MDA-MB-231, MDA-MB-330 and HBL-100), employing steroid concentrations of 1 nM. At this concentration, formation of ester sulphates was a major route of metabolism in the ER + cell lines; E2 yielding a mean of 6.5 pmol estrogen monosulphates/mg DNA in 16 h and ADIOL yielding a mean of 9.4 pmol C19-5-ene steroid monosulphates/mg DNA in 16 h. In three of the four ER - cell lines, formation of sulphates from E2 occurred at an eight-fold lower rate (mean 0.8 pmol estrogen sulphates/mg DNA in 16 h), whereas MDA-MB-330 cells did not form estrogen sulphates. Only one of the four ER- cell lines (BT-20) sulphurylated ADIOL and this was at a 12-fold lower rate compared to the mean value for the ER + cel lines. Oxidation of E2 and ADIOL occurred in all cell lines and was generally the major route of metabolism in the ER - cells. A significant correlation between formation of estrone and dehydroepiandrosterone occurred for all cell lines (r = 0.98, P less than 0.001) indicating that the same 17 beta-hydroxysteroid dehydrogenase was probably involved. Since ADIOL is estrogenic in a number of systems at the concentration found in the blood of Western women (approximately 2 nM), the coordinated expression of hydroxysteroid sulphotransferase, estrogen sulphotransferase, and ER, supports the concept of a functional relationship between estrogen action via ER and sulphurylation reactions.

17-Hydroxysteroid Dehydrogenases↗

PubMed1985

17 beta-Hydroxysteroid oxidoreductase: a ubiquitous enzyme. Interconversion of estrone and estradiol-17 beta in BALB/c mouse tissues.

A survey was conducted to define the sites of 17 beta-hydroxysteroid oxidoreductase activity in organs and tissues of male and female BALB/c mice, as well as the favored direction of the oxidoreductase reaction in intact tissues. The enzyme activity was assayed by use of radiolabeled estrone and estradiol-17 beta as substrates. Estrone formation from estradiol-17 beta was demonstrated in all tissues. The formation of estradiol-17 beta from estrone was demonstrated in most tissues, however, it was barely detected or was undetectable in the glandular stomach, small intestine, cecum, and large intestine. Thus, 17 beta-hydroxysteroid oxidoreductase activity is expressed in all BALB/c mouse organs and tissues. Approximately two-thirds of the tissues and organs examined, including those of the reproductive tracts, favored the conversion of estrone to estradiol-17 beta rather than the reverse reaction. The results of this study, however, represent qualitative estimates of 17 beta-hydroxysteroid oxidoreductase activity in BALB/c mouse tissues that are uncorrected for conversion to hydroxylated metabolites. These in vitro findings suggest that the 17 beta-hydroxysteroid oxidoreductase catalyzed reduction of estrone may contribute to the maintenance of physiologic levels of estradiol-17 beta in estrogen responsive tissues.

17-Hydroxysteroid Dehydrogenases↗

PubMed1980

Steroidal control of rat uterine 17 beta-hydroxysteroid dehydrogenase activity.

The interconversion of estradiol-17 beta and estrone in the rat uterus is due to the action of 17 beta-hydroxysteroid dehydrogenase. Whole uteri or 800 x g supernatant fractions of the uteri were incubated in the presence of [3H] estradiol-17 beta and NAD at 37 degrees C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17 beta and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17 beta-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17 beta, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17 beta-hydroxysteroid dehydrogenase activity of estradiol-17 beta treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17 beta administration. These results suggest that estradiol-17 beta caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17 beta-hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17 beta.

17-Hydroxysteroid Dehydrogenases↗

PubMed1987

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver.

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Inhibition of Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase by licorice-derived compounds and crystallization of an enzyme-cofactor-inhibitor complex.

Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase reduces the C20 ketone on glucocorticoids and progestins. We find that two licorice-derived compounds, glycyrrhizic acid and carbenoxolone, inhibit this enzyme with microM Kis. Inhibition is competitive, indicating that these compounds are binding at or close to the catalytic site. Carbenoxolone's high aqueous solubility and affinity for 3 alpha,20 beta-hydroxysteroid dehydrogenase enabled us to prepare crystals of a carbenoxolone-NADH-enzyme ternary complex, which preliminary X-ray analysis indicates has a crystal structure that is significantly different from that of the 3 alpha,20 beta-hydroxysteroid dehydrogenase-NADH complex. A comparison of the tertiary structures of these two complexes should prove useful in understanding this enzyme's catalytic mechanism, as well as those of two homologous enzymes, mammalian 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase that also are inhibited by carbenoxolone.

Binding Sites↗

PubMed1993

Inhibition of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase by antiestrogens.

Circulating estrone sulfate levels are 10-fold higher than the free estrone and estradiol levels in postmenopausal women and could form a reservoir from which the free estrogens could be synthesized in situ in breast cancer tissues. The enzymes catalyzing conversion of estrone sulfate to free estrone and estradiol are estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase, respectively. Selective blockade of these two enzymes may provide a means of reducing tumor estrogen levels and promoting tumor regression. The present study characterized the kinetics of several potential inhibitors of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase in vitro in rat breast tumors and compared these effects to those in human tissues. The antiestrogen ICI 164384 as well as tamoxifen and its metabolites inhibit estrone sulfatase via noncompetitive mechanisms at Kis ranging from 11-1130 microM in rat breast tumors. The steroid sulfates (pregnenolone sulfate and dehydroepiandrosterone sulfate) on the other hand, act as competitive inhibitors with Kis ranging from 4 to 6 microM. ICI 164384 and the tamoxifen metabolite 4-hydroxytamoxifen also blocked 17 beta-hydroxysteroid dehydrogenase at concentrations of 470 and 275 microM, respectively. In human breast tumors, 4-hydroxytamoxifen and desmethyltamoxifen blocked estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase but at higher concentrations than in the rat (i.e. IC50s of 1000-2000 microM). The inhibition caused by the antiestrogens requires concentrations at least 100-fold higher than those necessary for antiestrogenic effects. Although blockade of enzyme action is significant in vitro, and could also be in vivo, the effects of antiestrogens on enzyme inhibition are likely to be outweighed by their ability to block estrogen receptor-mediated effects in patients.

17-Hydroxysteroid Dehydrogenases↗

PubMed1993

3 beta-hydroxysteroid dehydrogenase deficiency in hyperandrogenism.

OBJECTIVE: Deficient adrenocortical 3 beta-hydroxysteroid dehydrogenase activity has been reported in 5% to 30% of hyperandrogenic women. Our objective was to determine the incidence and degree of 3 beta-hydroxysteroid dehydrogenase deficiencies in hyperandrogenism. STUDY DESIGN: A prospective study of adrenal function in patients with hyperandrogenism was performed in a tertiary care university medical center. Eighty-six consecutive patients with hirsutism or hyperandrogenic oligomenorrhea were studied; 26 healthy eumenorrheic women served as controls. All subjects underwent serum sampling at rest and a 1-hour adrenal stimulation test with 1 mg of intravenously corticotropin-(1-24). Dehydroepiandrosterone sulfate, androstenedione, sex hormone-binding globulin, total and free testosterone, and luteinizing and follicle-stimulating hormones were measured in basal serum; dehydroepiandrosterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone were measured in basal and corticotropin-stimulated serum. On the basis of experience with genetically defined 21-hydroxylase late-onset adrenal hyperplasia, patients were presumed to suffer from 3 beta-hydroxysteroid-deficient late-onset adrenal hyperplasia if they demonstrated a dehydroepiandrosterone or 17-hydroxypregnenolone response to corticotropin-(1-24) stimulation (absolute poststimulation level or net increment) greater than threefold the upper 95th percentile of controls. RESULTS: Three women of two families (2.3%) had a 17-hydroxyprogesterone response consistent with 21-hydroxylase-deficient late-onset adrenal hyperplasia and were excluded from further study. Eighteen (21%) of the remaining patients had a 17-hydroxypregnenolone poststimulation increment above the upper 95th percentile of controls (13.9 nmol/L), and two had an elevated dehydroepiandrosterone increment (> 19.5 nmol/L). However, no patient exceeded threefold the upper control limit for either steroid response. Patients with an exaggerated dehydroepiandrosterone or 17-hydroxypregnenolone increment had higher circulating dehydroepiandrosterone sulfate levels but similar basal total and free testosterone, sex hormone-binding globulin, luteinizing and follicle-stimulating hormone concentrations, basal or stimulated androstenedione, dehydroepiandrosterone/androstenedione, and 17-hydroxypregnenolone/17-hydroxyprogesterone than their less responsive counterparts. CONCLUSIONS: Although an exaggerated response of 17-hydroxypregnenolone to adrenal stimulation is common in hyperandrogenism, a response severe enough to merit consideration as 3 beta-hydroxysteroid dehydrogenase-deficient late-onset adrenal hyperplasia was not encountered in this unselected patient population, suggestive of the rarity of this disorder.

17-alpha-Hydroxypregnenolone↗

PubMed1991

Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase.

3 beta-Hydroxysteroid dehydrogenase/steroid isomerase has been purified to homogeneity from bovine adrenal glands. A single protein of molecular weight 42,090 +/- 40 containing both enzyme activities has been isolated. Approximately 86% of the amino acid sequence of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase has been obtained by sequencing peptides isolated from digests with trypsin and lysyl endopeptidase and by chemical cleavage with CNBr. The sequence obtained is identical with that of the deduced amino acid sequence of the bovine ovarian 3 beta-hydroxysteroid dehydrogenase/steroid isomerase [Zhao et al. (1989) FEBS Lett. 259, 153-157], with the exception that the N-terminal methionine residue found in the bovine ovarian sequence is not present in the mature bovine adrenal enzyme. On the basis of the primary structure and comparisons with other NAD+ binding proteins, we propose a structural model of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase localizing the NAD+ binding site as well as the membrane-anchoring segment.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

A 17 beta-hydroxysteroid dehydrogenase of female rabbit liver cytosol. Purification and characterization of multiple forms of the enzyme.

Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.

17-Hydroxysteroid Dehydrogenases↗