Leydig cells from neonatal pig testis abundantly express 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 and effectively inactivate cortisol to cortisone.

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) isozymes, designated types 1 and 2, catalyze the interconversion of physiologically active glucocorticoids and inactive 11-keto forms. The presence of types 1 and 2 was determined in neonatal pig testis and Leydig cells purified from testes by reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Type 2 mRNA was expressed at a high level in neonatal pig testis. In particular, in the entire testis, a higher level of type 2 was expressed compared to type 1. Furthermore, these expression levels in the testis were compared with the expression levels of the respective isozymes in pig liver and kidney, which are known to have high levels. Next, the direction of glucocorticoid metabolism in intact Leydig cells was examined, and only oxidation from cortisol to cortisone was detected. Virtually no reduction of cortisone to cortisol was detected. Using a microsomal enzyme preparation from Leydig cells, type 2 exhibited potent oxidation activity, and the activity was higher than the oxidation activity catalyzed by the type 1 isozyme. In kinetic analysis, the K(m) and V(max) for type 1 were 1.36 microM and 0.91 nmol/(h mg), respectively, and 0.38 microM and 1.25 nmol/(h mg), respectively, for type 2. The results of the present study using neonatal pig testis suggest that not only 11beta-HSD type 1 but also type 2, which is abundantly expressed, plays important roles in cortisol inactivation in pig Leydig cells, and furthermore, that excess cortisol will cause glucocorticoid-mediated suppression of testosterone production in even neonatal pig Leydig cells.

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion.

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.

Purification of leukocytosis-promoting substance from bovine parotid gland extract.

A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 . 10(4), and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.

Deletion of 12 carboxyl-terminal residues from pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase affects steroid metabolism.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase (3alpha/beta,20beta-HSD) is 80-85% identical to human, rat, and mouse carbonyl reductases. However, pig 3alpha/beta,20beta-HSD contains an extra 12 amino acids at its COOH-terminus that these other mammalian carbonyl reductases lack. We constructed a pig 3alpha/beta,20beta-HSD mutant, G278opal, which lacks these amino acids and found that compared to wild-type 3alpha/beta,20beta-HSD, G278opal has a 10-fold lower catalytic efficiency for testosterone and progesterone. G278opal also has lower 3alpha- and 20beta-reductase and increased 3beta-reductase activity compared to wild-type 3alpha/beta,20beta-HSD. Binding of NADPH to G278opal was similar to that of wild-type 3alpha/beta,20beta-HSD. The recently determined three-dimensional structure of 3alpha/beta,20beta-HSD, without a steroid substrate, shows the 12 COOH-terminal amino acids in a random configuration. Our data indicate that the 12 COOH-terminal amino acids have a role in steroid metabolism suggesting that binding of steroid to wild-type 3alpha/beta,20beta-HSD induces a conformational change in which the 12 COOH-terminal amino acids interact with the steroid substrate.

Circulating levels of isoflavones and markers of 5alpha-reductase activity are higher in Japanese compared with New Zealand males: what is the role of circulating steroids in prostate disease?

Epidemiological evidence implicates dietary isoflavone intake as protective against prostate disease. A putative mechanism is attenuated circulating androgen levels in male populations consuming an isoflavone rich diet. We investigated this hypothesis by collecting plasma from 60 Japanese and 60 New Zealand males aged between 21 and 31 years each consuming their traditional diets. We measured plasma testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), the combined levels of androsterone sulfate and epiandrosterone sulfate (AoS/epiAoS), sex hormone-binding globulin, and cortisol and corticosteroid-binding globulin as well as the isoflavones genistein and equol. Plasma genistein and equol levels were several times higher in Japanese males as would be expected from an isoflavone rich diet. However, androstenedione, DHEAS, calculated free testosterone and paradoxically markers of 5alpha-reductase, DHT and AoS/epiAoS were all also significantly higher in Japanese rather than the New Zealand male counterparts. All other comparisons were not significant. Plasma DHT and DHEAS correlated positively with plasma equol and plasma AoS/epiAoS correlated positively with genistein levels. Taken together the results suggest that, rather than reduced levels of steroidogenesis, Japanese males may have increased 5alpha-reductase activity and possibly altered 17beta OH steroid dehydrogenase activity. Significantly the positive association between isoflavones levels and 5alpha-steroids is counter-intuitive to isoflavone intake offering prostate protection, unless this is postulated to occur through other mechanisms.

Aromatase expression in the human fetal osteoblastic cell line SV-HFO.

A number of clinical studies have highlighted the importance of estrogen in bone growth and maintenance in men and postmenopausal women. In these instances, estrogen is synthesized locally within bone tissue by aromatase, encoded by the CYP19 gene. The mechanisms regulating aromatase expression in bone, however, are unclear. In this work we characterized the expression of aromatase activity and gene transcripts in the human fetal osteoblastic cell line, SV-HFO. Aromatase activity and gene transcript expression were stimulated by dexamethasone. Oncostatin M strongly stimulated aromatase expression in synergy with dexamethasone. These factors induced CYP19 transcripts that included the sequence of exon I.4 in their 5'UTR. Consistent with this, a reporter construct harboring the genomic sequence of the promoter region of exon I.4 (promoter I.4) was also activated by dexamethasone and oncostatin M. 5' deletion and mutation analysis revealed important roles for a glucocorticoid response element, an interferon gamma activating sequence and a putative binding site for Sp1. Transfection of exogenous glucocorticoid receptor, STAT3 or Sp1 increased promoter activity, indicating a potential role for these transcription factors in regulating aromatase expression in SV-HFO cells. These data suggest that the SV-HFO cell line is a valuable model with which to elucidate the mechanisms regulating local estrogen synthesis in osteoblasts.

Forskolin up-regulates aromatase (CYP19) activity and gene transcripts in the human adrenocortical carcinoma cell line H295R.

A number of conditions related to sex-reversal in boys and men and precocious puberty in girls are caused by estrogen-secreting adrenal tumors. In these tumors, cytochrome P450 aromatase (aromatase) that is encoded in the CYP19 gene is expressed at high levels. To investigate the molecular mechanism of aromatase expression in these adrenal tumors, we characterized the activity, gene transcript and genomic promoter region of aromatase in the human adrenocortical carcinoma cell line H295R. Aromatase activity and the transcript of the CYP19 gene were highly up-regulated by forskolin, but not by dexamethasone. The results from exon I-specific reverse transcriptase (RT)-PCR and the transfection of reporter constructs suggested that promoter I.3 and promoter II were activated in H295R. Deletion and mutation analysis suggested that cAMP response element-like sequence (CLS) and steroidogenic factor-1 (SF-1) motif, were critical for the activation of promoter II. The results of this work should provide the basis for the molecular analysis of aromatase expression in adrenocortical cells.

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: purification and some properties.

20 beta-Hydroxysteroid dehydrogenase was purified from a cytosol fraction of neonatal pig testes to homogeneity as demonstrated by polyacrylamide gel electrophoresis (PAGE) and by isoelectric focusing. The molecular weight was estimated to be 30,500 using PAGE with sodium dodecyl sulfate and the gel filtration method. Molecular estimations showed that the purified enzyme consisted of a single polypeptide chain. It catalyzed the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxypregn-4-en-3-one with NADPH. Furthermore, the C21-steroids, such as progesterone, pregnenolone, 17 alpha-hydroxypregnenolone, deoxycorticosterone, and deoxycortisol were also reduced by the purified enzyme. Apparent Km values for 17 alpha-hydroxyprogesterone, progesterone, pregnenolone, and deoxycorticosterone were 9.4, 1.5, 4.0, and 8.6 microM, respectively. The enzyme did not show 20 alpha-hydroxysteroid dehydrogenase activity. The maximum rate of enzyme activity was observed at 45 degrees C and optimum pH was at pH 5.5. The enzyme activity was strongly inhibited by heavy metal ions such as Hg2+ and Cu2+.

Evidence for involvement of cytochrome P-450-linked oxygenase system in the conversion of C21-steroids to delta 16-C19-steroids catalyzed by pig testicular microsomes.

Formation of androstadienone or androstadienol, a delta 16-C19-steroid, from progesterone or pregnenolone is catalyzed by the so-called delta 16-C19-steroid synthesizing enzyme in the pig testicular microsomes. The enzyme activity was also present in the testicular microsomes prepared from neonatal pig. The enzyme activity was considerably inhibited by CO, and such cytochrome P-450 inhibitors as SU 8000, SU 10603, and metyrapone. delta 16-C19-Steroid synthesizing enzyme activity was extracted from the testicular microsomes by sodium cholate in potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol, and the solubilized enzyme activity was partially purified by DEAE-cellulose column chromatography. It was shown by reconstitution of the enzyme activity that delta 16-C19-steroid synthesizing enzyme is a cytochrome P-450-linked oxygenase system dependent on cytochrome P-450-reductase and cytochrome b5. In particular, cytochrome b5 was an essential component for the activity of delta 16-C19-steroid synthesizing enzyme.

Inhibitory effect of some imidazole antifungal compounds on the synthesis of 16-ene-C19-steroid catalyzed by pig testicular microsomes.

The activity of the enzyme (16-ene-C19-steroid synthesizing enzyme) responsible for the conversion of C21-steroids to 16-ene-C19-steroids, which was localized on pig testicular microsomes, was inhibited by some typical imidazole antifungal compounds such as clotrimazole, econazole, miconazole and ketoconazole which are known to be universal inhibitors of cytochrome P-450-dependent enzymes. The 50% inhibitory concentrations of clotrimazole, econazole and miconazole were 0.29, 0.36 and 1.25 microM, respectively for 16-ene-C19-steroid synthesizing enzyme activity. Clotrimazole was the most powerful inhibitor of all the compounds examined, which shows the competitive inhibition for 16-ene-C19-steroid synthesizing enzyme activity. The Ki-value was 0.26 microM for its activity. The degree of the inhibition by these imidazole compounds was very similar to the inhibition of 17 alpha-hydroxylase and C17,20-lyase activities on pig testicular microsomes.

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme.

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.

Immunochemical distribution and immunohistochemical localization of 20beta-hydroxysteroid dehydrogenase in neonatal pig tissues.

Immunochemical distribution of 20beta-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20beta-HSD antibody. 20beta-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20beta-HSD were detected in the testis, followed by the kidney and liver, by the [125I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20beta-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20beta-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20beta-HSD has a very important physiological role in testicular function during the neonatal stage.

Direct expression of pig testicular 3 alpha/beta (20 beta)-hydroxysteroid dehydrogenase in Escherichia coli.

The cDNA coding for pig testicular 3 alpha/beta (20 beta)-hydroxysteroid dehydrogenase was expressed in Escherichia coli by placing it under the control of an isopropylthiogalactoside (IPTG) inducible tac promoter. Production of 3 alpha/beta (20 beta)-HSD was demonstrated by Western blotting and by catalytic activity with 5 alpha-dihydrotestosterone as a substrate for 3 alpha/beta-HSD, and progesterone and 17 alpha-hydroxyprogesterone as substrates for 20 beta-HSD in the presence of NADPH. The 3 alpha/beta (20 beta)-HSD enzyme was detected in a soluble fraction of the lysate of E. coli added to IPTG to induce the synthesis of the protein. Its molecular weight was estimated to be 30.5 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant 3 alpha/beta (20 beta)-HSD was purified to apparent homogeneity as determined by SDS-PAGE by column chromatography using DEAE-cellulose. The purified enzyme reduced not only steroids but also prostaglandins and other carbonyl compounds including aldehydes, ketones and quinones as demonstrated in native enzymes purified from pig testes. The amino terminus of the purified enzyme was serine which was coded next to the ATG start codon, and the sequence of the amino terminal 24 residues was identical with the coding amino acid in the cDNA; whereas, the amino terminus of the native 3 alpha/beta (20 beta)-HSD was not detected suggesting that the N-terminal amino acid was blocked.

Crystallization and preliminary X-ray diffraction studies of a mammalian steroid dehydrogenase.

20 beta-Hydroxysteroid dehydrogenase from the cytosolic fraction of neonatal pig testis is a NADPH-dependent enzyme that catalyzes the reduction of the C-20 ketone of C21-steroids. It is 85% homologous in amino acid sequence to the human enzyme, carbonyl reductase. The enzyme has been crystallized from 36% saturated ammonium sulfate in 10 mM 2-[N-Morpholino]ethanesulfonic acid buffer. The size and the quality of nicely formed square bi-pyramidal crystals were improved by using a "seeding" technique. The crystals diffract X-rays to at least 2.5 A resolution. The space group is P4(1)2(1)2 (or P4(3)2(1)2) and the unit-cell dimensions are a = b = 58.53 A, c = 165.64 A. There is one molecule (M(r) = 30.5 kDa; 289 amino acid residues) in the asymmetric unit. An intensity data set to 2.5 A has been collected with an overall Rmerge of 6.6% for all reflections.

Purification and characterization of 3 alpha/beta-hydroxysteroid dehydrogenase from mature porcine testicular cytosol.

NADPH-dependent 3 alpha/beta-hydroxysteroid dehydrogenase (3 alpha/beta-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. The molecular weight of the enzyme was estimated to be 31 kDa by SDS-polyacrylamide gel electrophoresis and 40 kDa by gel filtration chromatography indicating that the native 3 alpha/beta-HSD is a monomer. The isoelectric point of the purified enzyme was found to be 6.2 by density gradient isoelectric focusing and 6.4 by chromatofocusing. The enzyme reduced both 5 alpha- and 5 beta-DHT, 5 alpha- and 5 beta-dihydroprogesterone, 5 alpha- and 5 beta-dihydrocortisol, prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2 and 13,14-dihydro-15-keto-prostaglandin F2 alpha. Moreover, the enzyme caused rapid reduction of other carbonyl compounds including aldehydes, ketones and quinones. The rates of reduction of these compounds are fast relative to the rates of reduction of steroids and prostaglandins. The purified enzyme was inhibited by AgNO3, SH-reagent, quercetin, hexesterol, stilbestrol, disulfiram and divalent cation such as Cu2+, Hg2+ and Cd2+. The two enzymes show certain similarities (e.g. molecular weight, cross-reactivity to a common antibody) and certain striking differences (e.g. pI, effects of various inhibitors and greater enzyme activity towards steroids (neonatal form) or prostaglandins (mature form). Reasons are give for suggesting that these enzymes are closely related to carbonyl reductase.

Purification and characterization of aromatase from human placenta.

Aromatase from human placenta has been purified to homogeneity (MW 55,000). Enzymatic activity can be reconstituted with reductase from pig liver in an aqueous buffer or after incorporation of the enzyme into liposomes. In both cases the enzyme converts androstenedione to estrone and testosterone to estradiol. Aromatase shows a typical CO-spectrum when reduced with dithionite and a type I spectral shift with both substrates. The NH2 terminal amino acid sequence is hydrophobic but shows no homology to that of other cytochromes P-450. Five cysteine peptides have been isolated by HPLC following tryptic digestion of the [14C]-carboxymethylated protein. Amino acid sequences of these peptides reveal that histidine is the carboxy-terminal amino acid of the protein and that significant homology exists with corresponding peptides from other cytochromes P-450. Unique oligonucleotides (62 and 30 MER) synthesized on the basis of a 45 amino acid sequence near the center of the molecular have been used to clone the aromatase gene from a cDNA expression library from human placenta in lambda gt11.

Effect of phthalate esters and alkylphenols on steroidogenesis in human adrenocortical H295R cells.

We investigated the inhibitory effects of phthalate esters and alkylphenols on steroidogenesis by human adrenocortical H259R cells, a model of human steroidogenic cells. Dicyclohexyl phthalate (DCHP) at a concentration of 30 µM produced a significant decrease in the dibutyryl cAMP-stimulated cortisol secretion (76% reduction). 4-t-Pentylphenol (4-t-PP), 4-t-octylphenol (4-t-OP) and 4-nonylphenol (4-NP) also produced significant decreases in the dibutyryl cAMP-stimulated cortisol secretion by 58, 34 and 40%, respectively at 50 µM. Reductions in cortisol secretion by these chemicals were dose-dependent. To elucidate the inhibitory effects of DCHP, 4-t-PP, 4-t-OP and 4-NP on cortisol secretion from H295R cells, the effects of these chemicals on various steroidogenic enzymes, such as C(20,22)-lyase (CYP11A), 3ß-hydroxysteroid dehydrogenase type II (3ß-HSDII), 17α-hyroxylase/C(17,20)-lyase (CYP17), 21-hydroxylase (CYP21B) and 11ß-hydroxylase (CYP11B1), were investigated. DCHP significantly inhibited CYP21B activity at 25 µM. 4-t-OP strongly inhibited CYP11A activity at 12.5 and 25 µM, and inhibited CYP17 and CYP21B at 25 µM. Similarly, 4-NP inhibited CYP11A at 25 µM and strongly inhibited CYP17 and CYP21B at 12.5 and 25 µM. Neither DCHP nor any of the alkylphenols tested altered 3ß-HSDII activity.

[Purification of pig adrenal 3 beta-hydroxysteroid dehydrogenase/isomerase and influence on microsomal membrane destruction on its enzyme activity].

A 3 beta-hydroxysteroid dehydrogenase/delta 4-delta 5-isomerase (3 beta-HSD/isomerase) has been purified to homogeneity from the pig adrenal microsomes by solubilization with sodium cholate, followed by some conventional column chromatographies. The molecular weight of the enzyme was estimated to be 42000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km value for pregnenolone as the substrate of the purified enzyme was higher than that of the microsomal enzyme. The purified enzyme was less stable for heat treatment than the microsomal binding enzyme. The microsomal enzyme was treated with various agents or enzymes which destroyed the membrane structure. Lipid peroxidation with Fe2+, digestion with phospholipase A2 or C, and the addition of various free fatty acids, imidazole containing compounds and polymyxin B were utilized for the membrane destruction. Consequently, the 3 beta-HSD/isomerase activity was significantly reduced in all cases, and the Km value of the 3 beta-HSD/isomerase for pregnenolone increased. And the Vmax and Vmax/Km values significantly decreased. Therefore, it is strongly suggested that the normal membrane structure plays an important role in the maintenance of the 3 beta-HSD/isomerase activity.