SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Explore the source record for details and available documents.
The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.
Estradiol-17beta dehydrogenase activity in proliferative human endometrium (average of 1.5 nmole of estrone formed from estradiol/mg protein/h) was stimulated as much as as 6-fold during incubations of tissue slices in culture medium containing progesterone. Stimulation was already detectable at 7 h and the highest activity values were reached at 48-72 h of incubation in the presence of excess progesterone. Maximal stimulation was achieved with concentrations of the hormone of 0.25 mug/ml or higher. At concentrations approximately equal to midluteal plasma levels (20 ng/ml) more than 50% of the maximal response was observed. Norgestrel (17alpha-ethynyl-18-methyl-19-nortestosterone) was also effective in inducing enzymatic activity. The similarity of the effects obtained with progesterone (a possible substrate for estradiol dehydrogenase) and the synthetic progestin indicates that the stimulation of enzymatic activity was not due to substrate induction. Addition of estradiol to the culture medium had no influence on the activity of the enzyme. The induction of estradiol dehydrogenase by progesterone was inhibited by puromycin or actinomycin D. These observations indicate that progestational agents increase the rate of de novo synthesis of the enzyme. Stimulation of endometrial estradiol dehydrogenase was also observed after 2-3 day oral administration of medroxyprogesterone acetate to women in the follicular phase. In contrast, the enzymatic activity in endometrium obtained from women taking estrogens was found to be as low as in normal proliferative tissue. These in vitro and in vivo results point to progesterone as the agent responsible for the 10-fold increase in endometrial estradiol dehydrogenase activity observed during the luteal phase in menstruating women. Data obtained from superfusion studies of estrogen dynamics in endometrium indicate that changes in enzyme concentrations may play a physiologic role in the regulation of tissue levels of estradiol.
17-beta-Estradiol dehydrogenase is required for the enzymatic interconversion of estradiol and its weaker related sex steroid, estrone. We isolated and sequenced a complementary deoxyribonucleic acid clone for 17 beta-estradiol dehydrogenase from the BeWo choriocarcinoma cell line. Comparison of the BeWo complementary deoxyribonucleic acid sequence to a previously derived placental complementary deoxyribonucleic acid sequence yields greater than 98% homology. We also isolated the gene for 17 beta-estradiol dehydrogenase from the Jar human choriocarcinoma cell line and elucidated its primary nucleic acid structure. Significant differences in the Jar-deduced complementary deoxyribonucleic acid sequence clearly differentiate it from both the human placental and BeWo forms of 17 beta-estradiol dehydrogenase, indicating the existence of two genes for 17 beta-estradiol dehydrogenase in the human genome. Evaluation of 17 beta-estradiol dehydrogenase gene expression in BeWo and Jar cells was compared with expression in luteinized granulosa cells. Messenger ribonucleic acid for human placental 17 beta-estradiol dehydrogenase was identified in all three cell types as a 1.3 kilobase band on Northern blot analysis. A second messenger ribonucleic acid species measuring 2.1 kilobase was abundantly present in the granulosa cells. Whether these two species of messenger ribonucleic acid are involved in the regulation of the estradiol dehydrogenase genes is yet to be determined.
The inactivation of estradiol dehydrogenase by enzyme-generated 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one is accompanied by the formation of a lysine enaminone. The experiments leading to this conclusion involved degradation of the inactivated enzyme with Pronase and subsequent analysis by solution-state 13C NMR. The present paper reports solid-state 13C NMR experiments on lyophilized intact inactivated enzyme which are free from problems due to Pronase digestion. These experiments combine conventional cross-polarization and magic-angle spinning with selective irradiation of resonances arising from a 13C double label in the steroid. Magnetization transfer between neighboring 13C nuclei is used to simplify the spectra and to identify peaks due to label. The formation of cysteine and lysine Michael adducts of the enzyme is established by comparisons with chemical shifts of solid model adducts.
17 beta-Estradiol dehydrogenase (EC 1.1.1.62) catalyzes the interconversion of estradiol and estrone in human term placenta. We have raised a specific polyclonal antibody to this abundant placental enzyme to study its role in late pregnancy events and its molecular biologic characteristics. In this work the 17 beta-estradiol dehydrogenase antibody was used to isolate and sequence a complementary deoxyribonucleic acid clone encoding about 98% of the amino acid sequence of the 17 beta-estradiol dehydrogenase molecule. This sequence verifies previous sequence data on the molecule's steroid binding site and also localizes a putative nicotinamide adenine dinucleotide binding region similar to that of many other pyridine nucleotide-dependent dehydrogenases. Isolation of the complementary deoxyribonucleic acid for 17 beta-estradiol dehydrogenase expands our knowledge of the structure-function relationships of the enzyme and is a major step in our understanding of its biologic function in pregnancy.
Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.
The activity of 17 beta-estradiol dehydrogenase (E.C. 1.1.1.62) was measured, and its distribution in the subcellular fractions of bovine placenta was compared. Assay of activity was based on the formation of radioactive estrone from 17 beta[4(-14)C]-estradiol. Either NAD+ or NADP+ can serve as cofactor for the enzyme. The nuclear and microsomal fractions of the placental homogenate exhibited the highest specific enzymatic activities before and after treatment with Triton X-100. Electron micrographs of these two fractions prior to treatment with Triton X-100 showed satisfactory purity. 17 beta-estradiol dehydrogenase from bovine placenta exhibits a pH optimum of about 9.5-10.5, and is activated by 5 x 10(-6)M ZnCl2; comparable concentrations of CaCl2 and MgCl2 inactivate the enzyme. The apparent Michaelis constants, Km, for 17 beta-estradiol and NAD+ are 1.4 x 10(-6)M and 5.5 x 10(-5)M respectively. No 17 alpha-estradiol dehydrogenase activity was demonstrable when using 17 alpha-estradiol as substrate.
Immunohistochemical techniques were used to search for the presence of 17beta-estradiol dehydrogenase activity in human endometrial and placental tissues, with the use of antibodies raised against highly purified human placental 17beta-estradiol dehydrogenase. Sensitivity and specificity of the antibodies were documented by radioimmunoassay and immunodiffusion on cellulose acetate. Although staining was consistently demonstrated in the syncytiotrophoblast layer of term placentas, in both cytoplasm and nuclei, no immunohistochemical reaction was observed in endometrial samples. These results support the contention that placental 17beta-estradiol dehydrogenase is immunologically dissimilar from the endometrial enzyme.
Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, copurified from the cytosol fraction of human term placenta, were identically inactivated by 6 beta-bromoacetoxyprogesterone. This affinity alkylating steroid binds at the enzyme-active site (Km = 866 microM; Vmax = 0.073 mumol/min/mg). Enzyme inactivation by four concentrations of 6 beta-bromoacetoxyprogesterone (molar ratio of steroid to enzyme, 71/1 to 287/1) causes irreversible and time-dependent loss of both the 17 beta- and 20 alpha-activities according to first order kinetics and affirms that the alkylating steroid is an active site-directed inhibitor (KI = 2.7 X 10(-3) M; k3 = 1.6 X 10(-3) s-1). Affinity radioalkylation studies using 6 beta-[2'-14C]bromoacetoxyprogesterone indicate that 2 mol of steroid are bound to each mole of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 6 beta-bromoacetoxyprogesterone carboxymethylates cysteine (56%), histidine (22%), and lysine (8%) residues in the active site. These results are identical with those reported for 2-bromo[2'-14C]acetamidoestrone methyl ether radioalkylation of purified "17 beta-estradiol dehydrogenase." The parallel inactivation of 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase by 6 beta-bromoacetoxyprogesterone further shows that both activities reside at a single enzyme-active site. The radioalkylation profile supports our proposed model of one enzyme-active site wherein the bound progestin and estrogen substrates are inverted, one relative to the other.
Estradiol dehydrogenase (E2DH) is a well-known progesterone-dependent enzyme in human endometrium, and its induction has been proposed as a means to test hormonal sensitivity of endometrial carcinoma. While administration of progestins to some patients with endometrial carcinoma resulted in increased endometrial E2DH activity, efforts to induce this enzyme, in vitro, in these tumors have been unsuccessful. The reasons for such failure were investigated in the present study. Progesterone receptor (PR) concentrations and E2DH activities were simultaneously measured in proliferative and malignant endometria under organ culture conditions. Cytoplasmic PR concentrations were determined by Scatchard plot analysis of [3H]progesterone binding in fresh samples and in tissue explants incubated in nutrient medium at 37 degrees in a humidified 5% CO2 atmosphere for various periods of time. Parallel incubations of explants with and without 500 ng medroxyprogesterone acetate per ml were carried out for monitoring E2DH induction. In proliferative endometrium, the progesterone-specific binding sites remained stable during the culture periods, and the E2DH activities were stimulated severalfold by medroxyprogesterone acetate. In contrast, the PR concentrations in carcinoma explants were undetectable after a 24-hr period, and this was associated with a lack of increase in E2DH activity. These findings provide evidence that progestin-induced endometrial E2DH activity is a receptor-mediated phenomenon. In addition, these results demonstrate clearly that the ineffectiveness of progestin to induce E2DH in endometrial cancer specimens, in vitro, is related to the instability of PR under culture conditions. It is suggested that any experiment designed to follow effects of steroids on target tissues must take into account the stability of steroid receptors under in vitro conditions.
The conversion of estrone to 14,15-secoestratrien-15-ynes, inactivators of estradiol dehydrogenase from human term placenta, is described. The optically pure precursor 7-acetoxy-octahydro-2-phenanthrenecarboxylic acid methyl ester is prepared from estrone in five steps and 40% yield. The unsubstituted propargylic secoestratriene diol, a mechanism-based inactivator of estradiol dehydrogenase, and the corresponding acetylenic ketone, an affinity label inactivator of the same enzyme, arise from the phenanthrene ester in three and four steps. The propargylic secoestratriene diol also competes with [3H]estradiol for binding to calf uterus estrogen receptor and possesses weak uterotrophic activity.
Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, present in the cytosol fraction of term human placenta, were co-purified with a constant ratio of specific activities, approximating 100:1, respectively. The "pure enzyme" is a single band on sodium dodecyl sulfate disc gel electrophoresis. To evaluate whether catalysis of the estrogen and progestin substrates occurs at a single active site, alkylation studies using 16 alpha-bromoacetoxyprogesterone were designed. This affinity alkylating steroid binds at the enzyme-active site (km 256 microM; Vmax = 0.012 mumol/min/mg), inactivates the enzyme in an irreversible and time-dependent manner which follows pseudo-first order kinetics, and causes coincident loss of both the 17 beta- and 20 alpha-activities. Affinity radioalkylation studies using 16 alpha-[2'-3H]bromoacetoxyprogesterone indicate that 2 mol of steroid bind per mol of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 16 alpha-bromoacetoxyprogesterone dicarboxymethylates a histidyl residue in the active site. These results are identical with those reported for 16 alpha-[2'-3H]bromoacetoxyestradiol 3-methyl ether inactivation and radioalkylation of identically purified "17 beta-estradiol dehydrogenase." Computer graphics were used to construct a model in which: 1) binding of estrogen and progestin substrates at one active site permits stereospecific catalysis; 2) the estrogen and progestin analogs' alkylating side arms have access to a common histidine residue. These observations clearly demonstrate that the catalysis of estrogen and progestin substrates can occur at a single active site of one enzyme.
Two pyridine nucleotide linked oxidoreductase activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, which were copurified from human placental cytosol as a homogeneous enzyme preparation, may represent dual activity by one enzyme. The affinity labeling nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, which binds at the cofactor site as a competitive inhibitor of NADH (ki = 1.7 mM), simultaneously and identically inactivated both the 17 beta and 20 alpha activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. NADH and NAD+ markedly protected both activities from inactivation, and the substrate steroids, estrone, estradiol, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one, conferred similar protection, though less than cofactor, against simultaneous loss of both activities. Stoichiometric studies indicated that 2 mol of affinity labeling nucleotide were bound per mol of completely inactivated enzyme dimer. The coincident and identical loss of both activities under all experimental conditions is further evidence that 17 beta-estradiol dehydrogenase and 20 alpha-hydrosteroid dehydrogenase in human placental cytosol represent bifunctional, stereospecific, oxidoreductase activity at one active site on a single protein.
OBJECTIVE: 17 beta estradiol dehydrogenase (17 beta DH) is a model for pyridine-dependent steroid-converting enzymes. To define the structural and functional parameters of 17 beta DH, we created an expression system for production of abundant, homogeneous enzyme. METHODS: A full-length 17 beta DH cDNA clone was engineered into the inducible expression vector pMON 5839. After induction of plasmid-bearing Escherichia coli JM109 cells, the authenticity of the recombinant human placental 17 beta DH (r17 beta DH) was evaluated. RESULTS: Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17 beta DH with native human placental enzyme. The amino acid sequence, enzyme activity, Vmax, K(m), and kcat of r17 beta DH matched that of the native enzyme. CONCLUSION: Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17 beta DH without the expense and time commitment of baculoviral or eukaryotic cell lines. We are now able to use r17 beta DH and its mutants to elucidate the mechanisms of action of this class of enzyme.
Kinetics of estradiol dehydrogenase (E2DH) activity and the in vitro effects of progesterone (P) and synthetic steroids on E2DH activity were investigated in human normal endometrium and endometrial carcinoma. In proliferative and secretory endometrium and endometrial carcinoma, E2DH activities were 1.5 +/- 0.2, 10.1 +/- 1.1 and 1.2 +/- 0.1 nmol/mg protein/h (mean +/- SEM), Km was 2.3 microM, and Vmax were 0.20, 1.7 and 0.14 nmol/mg protein/10 min, respectively. Culturing proliferative endometria with progestogens resulted in a time- and dose-dependent stimulation of E2DH activity up to 72 h and 10(-6) M, respectively. Medroxyprogesterone acetate had the highest effect to stimulate E2DH activity among the steroids investigated. Chlormadinone acetate, norethindrone, P and R2323 were also effective. However, danazol, lynestrenol and E2 had negligible effect. Histological examination showed that progestogens caused early secretory change in the proliferative endometrium. These results indicate that the progestational activity is responsible for the elevation of E2DH activity in proliferative endometrium and that the extent to which each steroid increases E2DH activity may correlate with its local progestational activity. In the endometrial carcinoma, progestogens also stimulated E2DH activity in seven cases out of nine during culture for 48 h, but the elevation was lower than that in the proliferative endometrium.
Porcine 17 beta-estradiol dehydrogenase (EDH) was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the back reaction with NADPH. The 32 kDa EDH is cut from an 80 kDa primary translation product with a multidomain structure unknown for other hydroxysteroid dehydrogenases. The highest EDH activities and strongest immunoreactions are found in liver (hepatocytes) and kidney (proximal tubuli) followed by uterus (luminal and glandular epithelium), lung (bronchial epithelium). Progesterone treatment of ovariectomized gilts stimulates oxidative EDH activity in uterus, anterior pituitary, skeletal muscle (diaphragm) and kidney. Constitutive levels of EDH activity were seen in the adrenals, the lung and the liver.
Immunohistochemistry employing immunoglobulin G fractions raised against aromatase cytochrome P-450 and antiserum against 17 beta-estradiol dehydrogenase was used to localize these two steroid-converting enzymes in the human placenta. Immunostaining for both enzymes was found exclusively in the syncytiotrophoblast, while the underlying cytotrophoblast and the villus core did not stain. Ultrastructural examination of aromatase cytochrome P-450- and 17 beta-estradiol dehydrogenase-labeled sections disclosed immunoreactive product in the membranes of the endoplasmic reticulum; the nucleus, mitochondria, Golgi apparatus, and secretory granules were free of staining. These findings suggest that the syncytiotrophoblast is actively involved in the synthesis and metabolism of estrogens and in their role in placental endocrine function.