Charles H. Blomquist

17β-Hydroxysteroid and 20α-hydroxysteroid dehydrogenase activities of human placental microsomes: Kinetic evidence for two enzymes differing in substrate specificity☆

Abstract During storage at 4 °C, the 17β-hydroxysteroid dehydrogenase activity of human placental microsomes with estradiol-17β was more stable than that with testosterone. In order to evaluate the basis for this difference, kinetics with C18-, C19-, and C21-steroids as substrates and/or inhibitors was studied in conjunction with an analysis of the effects of detergents. Both 17β-hydroxysteroid dehydrogenase (17β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) activities were detected. At pH 9.0, apparent Michaelis constants were 0.8, 1.3, and 2.3 μ m for estradiol-17β, testosterone, and 20α-dihydroprogesterone, respectively. 17β-HSD activity with testosterone was inhibited by estradiol-17β,5α-dihydrotestosterone, 5β-dihydrotestosterone, 20α-dihydroprogesterone, and progesterone. In each case 90 to 100% inhibition was observed at 50 to 200 μ m steroid. Activity with 20α-dihydroprogesterone was similarly sensitive to inhibition by C19-steroids. By contrast, 25 to 45% of the activity with estradiol-17β was not inhibited by high concentrations of C19- or C21-steroids and differed from the 17β-HSD activity with testosterone and the major fraction of that with estradiol-17β by being insensitive to solubilization by detergent. These results are consistent with an association of two dehydrogenase activities with human placental microsomes. One recognizes C18-, C19-, and C21-steroids as substrates with comparable affinities. The second appears to be highly specific for estradiol-17β. The former activity may account for most if not all of the oxidation-reduction at C-17 of C19-steroids and at C-20 of C21-compounds at physiological concentrations by term placental tissue.

Kinetic analysis of enzymic activities: Prediction of multiple forms of 17β-hydroxysteroid dehydrogenase

An overview of the application of kinetic methods to the delineation of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17 beta-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17 beta-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17 beta-HSD type 1 is a cytosolic enzyme highly specific for C18 steroids such as 17 beta-estradiol (E2) and estrone (E1). 17 beta-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E2 and E1. Useful parameters for the detection of multiple forms of 17 beta-HSD appear to be the E2/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17 beta-HSD type 2. The 17 beta-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17 beta-HSD which differ from 17 beta-HSD type 1 and type 2 in their kinetic properties.

Metabolism of estrogens and androgens by scleractinian corals.

Estrogens and androgens are steroids that act as reproductive hormones in vertebrates. These compounds have also been detected in reef-building corals and other invertebrates, where they are hypothesized to act as bioregulatory molecules. Experiments were conducted using labeled steroid substrates to evaluate metabolism of estrogens and androgens by coral homogenates. GC-MS analysis of 13C-labeled steroids showed that Montipora capitata coral homogenates or fragments could convert estradiol to estrone and testosterone to androstenedione and androstanedione, evidence that M. capitata contains 17beta-hydroxysteroid dehydrogenase and 5alpha-reductase. When homogenates from three coral species and symbiotic dinoflagellates (zooxanthellae) were incubated with tritiated steroid substrates, metabolites separated by thin-layer chromatography confirmed that 17beta-hydroxysteroid dehydrogenase activity occurred in all species tested. NADP+ was the preferred cofactor in dehydrogenation reactions with coral homogenates. Reduction of estrone and androstenedione occurred at lower rates and aromatization of androgens was not observed. It is unclear whether estrogens detected previously in coral tissues are produced endogenously or sequestered in coral tissue from dietary or environmental sources. Previous studies have demonstrated that corals can take up estrogens from the water column overlying coral reefs. Considered in total, these observations suggest corals could alter the concentration or form of steroids available to reef organisms.

The effects of betamethasone on fetal development in the rabbit

Pregnant rabbits were injected with either 0.8 mg of betamethasone or with a comparable volume of saline on days 24 and 25 of gestation and delivered by cesarean section on day 26. There was a significant reduction in the weight of fetuses and in the weights of fetal brain, lungs, liver, and placenta in betamethasone-treated animals. With respect to the fetal brain, the concentration of DNA, the average of cell number, cell size, cell weight, and cell phospholipids were unaffected whereas the concentrations of phospholipids and protein were elevated in the treatment group.

Steroid metabolism in cnidarians: Insights from Nematostella vectensis

Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnidarians contain a diverse complement of steroids, sterols, and other lipid metabolites, relatively little is known of the endogenous steroid metabolism or function in cnidarian tissues. Incubations of cnidarian tissues with steroid substrates have indicated the presence of steroid metabolizing enzymes, particularly enzymes with 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. Through analysis of the genome of the starlet sea anemone, Nematostella vectensis, we identified a suite of genes in the short chain dehydrogenase/reductase (SDR) superfamily including homologs of genes that metabolize steroids in other animals. A more detailed analysis of Hsd17b4 revealed complex evolutionary relationships, apparent intron loss in several taxa, and predominantly adult expression in N. vectensis. Due to its ease of culture and available molecular tools N. vectensis is an excellent model for investigation of cnidarian steroid metabolism and gene function.

Androgenic and estrogenic 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms

17beta-Hydroxysteroid dehydrogenase/17-ketosteroid reductases (17HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although 17HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, 17HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-17beta/testosterone (E(2)/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal 17HSD/KSR2 and cytosolic 17HSD/KSR5, isoforms reactive with both E(2) and T, with evidence of estrogenic 17HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated 17HSD/KSR5 was the predominant isoform. The presence of 17HSD/KSR5, which also has both 3alpha-HSD/KSR and 20alphaHSD/KSR activity, and 17HSD/KSR2 which also has 20alpha-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of 17β-hydroxysteroid oxidoreductase type 1 and type 2 activity of cytosol and microsomes from human term placenta, ovarian stroma and granulosa-luteal cells

A large body of evidence suggests multiple forms of 17 beta-hydroxysteroid oxidoreductase (17-HOR) regulate estrogen and androgen levels within gonadal and peripheral tissues. Two kinetically-differing 17-HOR activities have been detected in placental homogenates. 17-HOR type 1, found mainly in the cytosol, is highly reactive with estradiol-17 beta (E2) and estrone (E1) but not testosterone (T) (high E2/T activity ratio). Microsomal 17-HOR type 2 is reactive with both E2 and T (low E2/T activity ratio). In this study, 17-HOR activity of cytosol and microsomes from term placenta, ovarian stroma and granulosa-luteal cells was assayed under conditions which specifically differentiate between the two forms of the enzyme. Placenta had the highest activity with either E2 or T in both cytosol and microsomes and stroma the lowest. The highest specific activity with E2 and E1 was cytosolic in all samples. The highest specific activity with T was microsomal in placenta and ovarian stroma. E2/E1 activity ratios were comparable for cytosol and microsomes while E2/T activity ratios were comparable for placenta and stroma, but markedly elevated in granulosa-luteal (G-L) cell cytosol and microsomes. The results indicate trophoblast and ovarian stroma have more 17-HOR type 2 relative to type 1. G-L cells, in contrast, are relatively enriched in 17-HOR type 1 and thus have a greater capacity for net conversion of E1 to E2 under physiologic conditions. These differences may contribute to increasing serum and follicular fluid E2/E1 ratios during development of the dominant follicle.

Modification of the kinetic properties of 5-ene, 3β-hydroxysteroid dehydrogenase of human placental microsomes by hydrogen peroxide and 2-mercaptoethanol

The kinetic properties of 5-ene,3β-hydroxysteroid dehydrogenase of human placental microsomes were modified by H2O2 and 2-mercaptoethanol. With microsomes prepared in the presence of 2-mercaptoethanol the KM values were 0.035 μM for pregnenolone and 1.9 μM for NAD+. When the microsomes were treated with H2O2 the values were 1.20μM for pregnenolone and 30.0 μM for NAD+. The H2O2 effect was reversed by 2-mercaptoethanol. Identical effects were seen with detergent-solubilized enzyme. With H2O2-treated enzyme progesterone, androstenedione and 20α-hydroxyprogesterone were non-competitive inhibitors with KI values of 5.5, 0.83 and 4.1 μM respectively. The results indicate that the enzyme can exist in at least two kinetically different forms and that the km values for NAD+ and pregnenolone in vivo may be significantly lower than previously thought.

The molecular weight and substrate specificity of 20 β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans☆

Abstract The molecular weight of 20β-hydroxysteroid dehydrogenase was 111,000 when determined by agarose gel fitration and 106,000 by density gradient centrifugation. From gel electrophoresis in sodium dodecyl sulfate, after treatment with urea and 2-mercaptoethanol, the molecular weight was 27,000, consistent with the native molecule containing four subunits. After gel electrophoresis at pH 8.1, a single band was detected which stained for protein and activity with 5α-pregnan-20β-ol-3-one and 5α-androstan-3α,17β-diol. 20β-hydroxysteroid dehydrogenase was inactivated at pH 4.5 and the time course of inactivation was independent of the steroid used for activity measurements. Steroid substrates did not protect 20β-hydroxysteroid dehydrogenase against acid inactivation or affect enzyme fluorescence. It was concluded that the activity observed with the two substrates occurred at the same active center and that under the experimental conditions little steroid was bound to the enzyme in the the absence of coenzyme.