PubMed Health⌕ Search

Biomedical subjects

F Labrie

Publications and source records attributed to F Labrie.

At least 55 records · Page 3Linked to original sources
PubMed1992

Expression of human 17 beta-hydroxysteroid dehydrogenase in mammalian cells.

The insert of 1278 bp containing the entire coding region of cDNA encoding human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17 beta-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol are interconverted at a lower rate. The present data suggest the existence of two 17 beta-HSDs.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Serum prostate specific antigen as pre-screening test for prostate cancer.

Prostate cancer has become the most common cancer and the second cause of death due to cancer in men in North America. Since curative therapies are limited to early stages of the disease, the availability of an efficient, easy to perform, widely acceptable and cost-effective method of early detection of prostate cancer is particularly important. Thus, digital rectal examination, transrectal ultrasonography of the prostate as well as measurements of serum prostate specific antigen (PSA) were performed independently in a series of 1,002 men between 45 and 80 years old randomly selected from the electoral rolls of Quebec City and its vicinity as part of a screening program for prostate cancer. Using this population of randomly chosen men, various cutoff serum PSA values were selected in an attempt to find the optimal decision threshold that would indicate a much greater risk of having prostatic cancer. At a threshold value of 3.0 micrograms./l. the sensitivity and specificity of the test are 80.7 and 89.6%, respectively, while the area under the receiver operating characteristic curve reflecting the accuracy of the test is 87.8 +/- 3.3% (plus or minus standard deviation). Moreover, the negative predictive value was estimated at 98.6%, thus leaving only a 1.4% chance of missing cancer when the serum PSA value was 3.0 micrograms./l. or less. Most importantly, such a threshold level of serum PSA retains only 19% of the whole cohort as candidates for transrectal ultrasonography and expensive diagnostic procedures, thus leading to the finding of 1 prostate cancer of 4 such examinations. The present data indicate that simple measurement of serum PSA can be used efficiently as a pre-screening test for prostate cancer in the general population to identify, at a low cost, the subpopulation of men at a much greater risk of having prostate cancer, and who should then be submitted to the more elaborate and expensive diagnostic procedures.

Age Factors↗
PubMed1992

Congenital adrenal hyperplasia due to point mutations in the type II 3 beta-hydroxysteroid dehydrogenase gene.

Classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) deficiency is an autosomal recessive form of congenital adrenal hyperplasia characterized by a severe impairment of steroid biosynthesis in both the adrenals and the gonads. We describe the nucleotide sequence of the two highly homologous genes encoding 3 beta-HSD isoenzymes in three classic 3 beta-HSD deficient patients belonging to two apparently unrelated pedigrees. No mutation was detected in the type I 3 beta-HSD gene, which is mainly expressed in the placenta and peripheral tissues. Both nonsense and frameshift mutations, however, were found in the type II 3 beta-HSD gene, which is the predominant 3 beta-HSD gene expressed in the adrenals and gonads, thus providing the first elucidation of the molecular basis of this disorder.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyses an obligatory step in the biosynthesis of all classes of hormonal steroids, namely, the oxidation/isomerization of 3 beta-hydroxy-5-ene steroids into the corresponding 3-keto-4-ene steroids in gonadal as well as in peripheral tissues. Because humans are unique with some primates in having adrenals that secrete large amounts of the steroid precursors dehydropiandrosterone (DHEA) and its sulfate (DHEA-S) and its exceptionally large volume makes the skin an important site of steroid biosynthesis, we have isolated and characterized cDNA clones encoding 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase from a human skin lambda gt11 library. The longest clone obtained contains the entire coding sequence for type I 3 beta-HSD (372 amino acids) as well as an additional 131 nucleotides in the 5'-untranslated region. The insert of 1647 bp containing the entire coding region has been inserted in a pCMV expression vector and transfected into human cervical carcinoma cells (HeLa). The expressed enzyme efficiently catalyzes the transformation of pregnenolone, DHEA, and dihydrotestosterone into progesterone, 4-androstenedione, and 5 alpha-androstane-3 beta, 17 beta-diol, respectively. Using the enzyme expressed in HeLa cells, we have shown cyproterone acetate, a progestin used in the treatment of acne and hirsutism, as well as norgestrel and norethindrone, two steroids widely used as oral contraceptives, to be relatively potent inhibitors, with Ki values of 0.38 microM, 1.3 microM, and 1.2 microM, respectively. Immunohistochemical localization of 3 beta-HSD, illustrated by using an antibody raised against human placental 3 beta-HSD, shows that the enzyme is localized in sebaceous glands.

Amino Acid Sequence↗
PubMed1992

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Additive stimulatory action of glucocorticoids and androgens on basal and estrogen-repressed apolipoprotein-D messenger ribonucleic acid levels and secretion in human breast cancer cells.

Recent elucidation of the amino acid sequence of the progesterone-binding protein GCDFP-24, the major protein found in human breast gross cystic disease fluid, reveals that this glycoprotein corresponds to apolipoprotein-D (apo-D), a member of the alpha 2-microglobulin superfamily which binds small hydrophobic ligands. The present study describes the multiple hormonal control of apo-D mRNA levels, intracellular protein content, as well as secretion compared to cell proliferation in human ZR-75-1 breast cancer cells. In these cells, exposure to the synthetic glucocorticoid dexamethasone (DEX) markedly decreases basal as well as 17 beta-estradiol (E2)-induced cell proliferation while causing a maximal 10-fold stimulation of apo-D secretion in the presence or absence of E2. Incubation with 500 nM DEX or 10 nM dihydrotestosterone (DHT), alone and in combination, markedly increased apo-D mRNA/actin mRNA ratios by 16-, 22-, and 28-fold, respectively. Exposure to 1 nM E2 decreased the apo-D mRNA/actin mRNA ratio by 65%. In E2-treated cells, simultaneous exposure to DHT, DEX, and DHT plus DEX markedly increased the apo-D mRNA/actin mRNA ratios by 50-, 35-, and 105-fold, respectively. The stimulatory effect of DEX on intracellular apo-D content and secretion was also additive to that of the androgen DHT in the presence or absence of E2. The present study provides the first data describing the hormonal regulation of apo-D mRNA levels and intracellular protein content and demonstrates the effect of glucocorticoids alone as well as their interaction with androgens and estrogens on these parameters as well as on apo-D secretion. As shown in the present data, the effects of steroids on apo-D gene expression, intracellular apo-D protein content, and secretion are opposite their respective specific effects on cell proliferation in human ZR-75-1 breast cancer cells.

Actins↗
PubMed1992

Structure of human type II 5 alpha-reductase gene.

The best known activity of steroid 5 alpha-reductase is the transformation of testosterone into dihydrotestosterone, the most potent androgen. Two types of human steroid 5 alpha-reductase cDNAs and the type I gene have previously been isolated and characterized. This report describes the isolation and characterization of the human type II 5 alpha-reductase gene, the gene most likely responsible for male pseudohermaphroditism due to 5 alpha-reductase deficiency as well as the one presumed to be involved in a major androgen-related diseases such as prostate cancer and benign prostatic hyperplasia. The type II 5 alpha-reductase gene contains five exons of 352, 164, 102, 151 and 1695 bp, respectively, which share 43.8% to 64.1% homology with exons of the corresponding type I gene. These exons are separated by four introns of greater than 29, and approximately 2.3, 2.0 and 3.0 kb. Analysis of primer extension products by polyacrylamide gel electrophoresis as well as by subcloning and sequencing reveals a start site located 71 nucleotides upstream the ATG initiating codon.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Regulation of 3-ketosteroid reductase messenger ribonucleic acid levels and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase activity in rat liver by sex steroids and pituitary hormones.

We have recently characterized three types of complementary DNA clones encoding predicted isoenzymes of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. Transient expression in nonsteroidogenic cells reveals that the type III isoenzyme specific for male liver does not display oxidative activity for classical substrates of 3 beta-HSD, in contrast to the two other 3 beta-HSD isoenzymes, thus showing exclusively 3-ketosteroid reductase (3-KSR) activity. In order to better understand the sex-specific control of 3 beta-HSD activity and type III 3-KSR gene expression in rat liver, we have studied in adult animals of both sexes the effect of sex steroids and hypophysectomy, pituitary implants, PRL, and GH on type III 3-KSR messenger RNA (mRNA) levels and 3 beta-HSD/delta 5-delta 4 isomerase activity as measured by the conversion of [14C]dehydroepiandrosterone into [14C] delta 4-androstenedione. Ribonuclease protection assay using types I-, II-, and III-specific complementary RNA probes reveals that type III transcripts are the only species detectable in liver RNA extracted from intact males, whereas no hybridization signal was detectable with any of the three probes in intact female liver RNA. In males, 15 days after castration, liver type III 3-KSR mRNA levels decreased by 80% compared to intact controls, whereas 3 beta-HSD activity was reduced by 48%. Administration of dihydrotestosterone (DHT) increased by 8.25-fold type III 3-KSR mRNA concentration and completely reversed the inhibitory effect of orchiectomy on 3 beta-HSD activity. In ovariectomized animals, treatment with DHT markedly increased type III 3-KSR mRNA accumulation and 3 beta-HSD activity, thus leading to values similar to those measured in intact males. Simultaneous treatment with 17 beta-estradiol almost completely abolished the stimulatory effect of DHT in female rats, whereas no significant effect was seen in males. Twenty-four days after hypophysectomy, type III 3-KSR mRNA levels were decreased by 50-55% in males, whereas in females these transcripts markedly increased from undetectable to 28-36% of the value measured in intact male rats. Treatment with DHT or 17 beta-estradiol for a period of 9 days starting 15 days after hypophysectomy had no effect in male and female rats. On the other hand, treatment with ovine PRL (1 mg, twice daily) had no effect in males but completely blocked the elevation of type III 3-KSR mRNA levels and 3 beta-HSD activity observed after hypophysectomy in females.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Immunocytochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human ovary.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid delta 5----4-ene-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain more information about the age-specific expression of 3 beta HSD in the human ovary, we have localized this enzyme by immunocytochemistry at the light microscopic level during fetal and postnatal periods of development in the human. Immunocytochemical localization was achieved using specific polyclonal antibodies developed against purified human placental 3 beta HSD. In the fetal ovary of 28-34 weeks, specific immunostaining for 3 beta HSD was exclusively detected in thecal cells surrounding primary follicles and in interstitial cells. From birth until puberty, no significant immunostaining for 3 beta HSD could be observed, while from puberty to menopause, staining was detected in theca interna cells as well as granulosa cells of growing follicles. The intensity of staining in the theca interna cells was always much higher than that in granulosa cells. Immunostaining was also found in the cytoplasm of luteinized granulosa and theca interna cells of the corpus luteum. Interestingly, there was an absence of immunoreactivity for 3 beta HSD in one to several layers of theca interna cells lying just beneath the basement membrane. These negative cells may correspond to fibroblast-like cells that are devoid of 3 beta HSD activity. In postmenopausal ovaries, immunolabeling was only found in dispersed interstitial cells, thus suggesting that the ovaries of postmenopausal women might be involved in sex steroid hormone secretion.

Aged↗
PubMed1992

Mechanisms of androgen production in male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency.

17 beta-Hydroxysteroid dehydrogenase (17 beta HSD) deficiency is a rare cause of male pseudohermaphroditism, but is a frequent disorder among a highly inbred Arab population in the Gaza strip. Affected individuals are born and reared as females until puberty, when marked virilization occurs, leading in many cases to the spontaneous adoption of a male gender role. To investigate the mechanisms and site(s) of androgen production, we determined the gonadal and extragonadal steroid patterns in two postpubertal male pseudohermaphroditism patients, who were castrated and reared as females. Before castration, both patients had very high plasma levels of androstenedione (delta 4-A), normal or moderately low levels of testosterone (T), and significantly elevated delta 4-A/T ratios (P less than 0.01). Dihydrotestosterone (DHT) levels were normal or high, while the DHT/T ratios were lower than normal (P less than 0.01), suggesting enhanced 5 alpha-reductase activity. These abnormalities were much more severe in spermatic venous blood. 17 beta HSD deficiency was also found in the delta 5-pathway, by high dehydroepiandrosterone (DHEA) levels and very high dehydroxyepiandrosterone/delta 5-androstenediol (DHEA/delta 5-diol) ratios, and in peripheral tissue metabolites, by very high androsterone glucuronide/3 alpha-androstanediol glucuronide ratios (P less than 0.01). The estrogen pathway was also impaired (P less than 0.01), even though both estrone and estradiol levels were elevated. Gonadectomy significantly reduced all androgens and estrogens (P less than 0.01), but when compared to 42 castrated controls, both patients had lower delta 4-A and higher T levels. The delta 4-A/T ratio was lower than that in controls, indicating normal to enhanced extragonadal 17 beta HSD activity. A similar pattern was observed in the delta 5- and estrogen pathways. DHT levels were within normal limits, and 3 alpha-diol was moderately decreased. These data suggest that testicular 17 beta HSD activity is under a different genetic control from that in extragonadal tissues. Affected males lack the testicular enzyme, but their extragonadal 17 beta HSD activity is normal or enhanced. Together with enhanced 5 alpha-reductase activity, this represents a highly efficient compensatory mechanism for androgen and estrogen production after puberty.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Immunohistochemical localization of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5----delta 4 isomerase in human placenta and fetal membranes throughout gestation.

The regulation of steroid production by the placenta and fetal membranes is important for both the maintenance of pregnancy and the timing of parturition. 3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5----delta 4-isomerase (3 beta HSD) catalyzes an obligatory step in the biosynthesis of steroid hormones. We have determined the localization of 3 beta HSD in the human placenta, fetal membranes, and umbilical cord throughout gestation by immunohistochemical analysis, using a polyclonal antibody raised in rabbits against a purified preparation of human placental 3 beta HSD. In placenta, immunoreactive (IR-) 3 beta HSD was localized in the syncytiotrophoblast and intermediate trophoblast cells at both villous and extravillous sites, but not in cytotrophoblast cells from 6 weeks gestation to term. At 6-7 weeks gestation, IR-3 beta HSD was distributed in the cytoplasm of syncytiotrophoblast in about half of placental villi. By 12-14 weeks, the syncytiotrophoblast of all placental villi stained positively for 3 beta HSD. In the fetal membranes, strong IR-3 beta HSD staining was found in the trophoblast and reticular layers of chorion and in invasive trophoblast cells in decidua, and weakly in decidual stromal cells and amniotic epithelium. No IR-3 beta HSD was found in amnion on the placental plate, but in the umbilical cord, IR-3 beta HSD was present in the amniotic epithelium and also in fibroblast cells in Warton's jelly. These observations demonstrate that the localization of 3 beta HSD immunoreactivity and, therefore, the presumed sites of delta 5- to delta 4-steroid interconversion throughout gestation are principally the syncytiotrophoblast and intermediate trophoblast cells in placenta and the trophoblast cells in chorion and decidua in fetal membranes.

Extraembryonic Membranes↗
PubMed1992

Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates.

Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1992

Androgens and breast cancer.

We have recently demonstrated that physiological levels of androgens exert direct and potent inhibitory effects on the growth of human breast cancer ZR-75-1 cells in vivo in nude mice as well as in vitro under both basal and estrogen-stimulated conditions. The inhibitory effect of androgens has also been confirmed on the growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. Such observations are in close agreement with the clinical data showing that androgens and the androgenic compound medroxyprogesterone acetate (MPA) have beneficial effects in breast cancer in women comparable to other endocrine therapies, including tamoxifen. Although the inhibitory action of androgens on cell proliferation in estrogen-induced ZR-75-1 cells results, in part, from their suppressive effect on expression of the estrogen receptor, the androgens also exert a direct inhibitory effect independent of estrogens. Androgens cause a global slowing effect on the duration of the cell cycle. These observations support clinical data showing that androgenic compounds induce an objective remission after failure of antiestrogen therapy as well as those indicating that the antiproliferative action of androgens is additive to that of antiestrogens. We have also recently demonstrated in ZR-75-1 human breast cancer cells the antagonism between androgens and estrogens on the expression of GCDFP-15 and GCDFP-24 which are two major proteins secreted in human gross cystic disease fluid. The effects of androgens and estrogens as well as those of progestins and glucocorticoids on GCDFP-15 and GCDFP-24 mRNA levels and secretion are opposite to those induced by the same steroids on cell growth in ZR-75-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1991

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells.

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e., dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Androgens↗
PubMed1991

Evidence for distinct dehydrogenase and isomerase sites within a single 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein.

Complementary DNA encoding human 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3 beta-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3 beta-HSD protein. In addition, using 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta, 17 beta-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

Androgen Antagonists↗
PubMed1991

Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells.

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase (3 beta-HSD) catalyzes the conversion of delta 5 -3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus representing an essential step in the biosynthesis of all classes of hormonal steroids. We have recently characterized two types of cDNA clones encoding rat 3 beta-HSD proteins, the rat type I protein being much more active than type II. In order to characterize further the functional difference between these two 3 beta-HSD types, transient expression of type I and type II 3 beta-HSD cDNAs was performed in HeLa human cervical carcinoma cells. The present study demonstrates that the type I 3 beta-HSD protein has a relative specificity 64- and 46-fold higher than type II protein for pregnenolone (PREG) and dehydroepiandrosterone (DHEA) as substrates, respectively. The Km values of type I and type II enzymes were calculated at 0.74 and 14.3 microM, respectively, using PREG as substrate whereas the respective Km values were 0.68 and 12.9 microM when DHEA was used, thus showing that their different relative specificity results largely from a different affinity for substrates. Since the change of 4 amino acid residues in type II could prevent the formation of a putative membrane-spanning domain (MSD) predicted between amino acid residues 75 and 91, chimeric cDNAs containing either type I MSD in type II (II + MSD) or an absence of this MSD in type I (I-MSD) were constructed and transiently expressed. The addition of MSD intype II 3 beta-HSD markedly increased the affinity leading to Km values similar to those found in type I 3 beta-HSD, namely 0.36 and 0.40 microM for PREG and DHEA, respectively. II + MSD chimera thus encodes a protein having a relative specificity for PREG and DHEA of 58 and 73%, respectively, to that of native type I 3 beta-HSD. Moreover, removal of MSD in the type I protein (I-MSD chimera) decreased the relative specificity of type I 3 beta-HSD protein for PREG and DHEA to only 0.37 and 0.48%, with respective Km values of 11.7 and 11.0 microM, thus strongly indicating the functional importance of this putative MSD which is predicted in wild type rat type I as well as in macaque and human 3 beta-HSD proteins.

Amino Acid Sequence↗