Masato Shinoda

Cytochrome b5 promotes the synthesis of Δ16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis☆

Conversion of progesterone to 17 alpha-hydroxyprogesterone plus androstenedione (17 alpha-hydroxylation) and to androstadienone (delta 16 synthetase activity) by microsomes from neonatal pig testis, were both inhibited by antibodies raised against homogeneous cytochrome P-450 C21 side-chain cleavage. Inhibition of the two activities showed the same relationship to the concentration of antibody added. Analogous results were obtained with pregnenolone as substrate. In a reconstituted enzyme system consisting of the homogeneous cytochrome P-450 C21 side-chain cleavage enzyme, P-450 reductase and NADPH, addition of cytochrome b5 resulted in the synthesis of the corresponding delta 16-C19-steroid from progesterone (androstadienone) and pregnenolone (androstadienol). The effect of cytochrome b5 was concentration-dependent and prevented by anti-cytochrome b5. It is concluded that the cytochrome P-450 C21 side-chain cleavage enzyme from pig testicular microsomes is also capable of synthesizing delta 16-C19-steroids and is, therefore, likely to be responsible for the large amounts of the pherormone androstadienone produced by male pigs.

Amino terminal sequence analysis of human placenta aromatase

The amino acid composition and the amino-terminal amino acid sequence from position 1 to 21 of human placenta aromatase were determined. In addition, a cysteine containing peptide with a sequence homologous to those of peptides containing the cysteine residue which was suggested to provide the proximal thiolate ligand to the heme in other cytochrome p-450 isozymes, was identified. The results indicate that aromatase is a cytochrome p-450 protein, probably derived from a new cytochrome p-450 family.

20β-hydroxysteroid dehydrogenase of neonatal pig testis: 3α/β-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

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

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3α/β-, 20α- and 20β-): Evidence for two forms of 3α/β-hydroxysteroid dehydrogenase

Abstract Three enzymatic activities (α/β-hydroxysteroid dehydrogenase, 20β- and 20α-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20β-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3α/β-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787–794]. We report here that neonatal pig testis is rich in 3α/β- and 20β-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of α/β-reduction rises to high levels whereas 20β-reduction remains low. Activity of 20α-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 (3α/β-plus 20β-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 3α/β-hydroxysteroid dehydrogenase without 20β-reductase activity. The possible functional significance of these changes is considered.

Immunochemical distribution and immunohistochemical localization of 20β-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 Henles 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.

Purification and characterization of 3α/β-hydroxysteroid dehydrogenase from mature porcine testicular cytosol

Abstract NADPH-dependent 3α/β-hydroxysteroid dehydrogenase (3α/β-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5α-dihydrotestosterone (5α-DHT) to both 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-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 3α/β-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 5α- and 5β-DHT, 5α- and 5β-dihydroprogesterone, 5α- and 5β-dihydrocortisol, prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2 and 13,14-dihydro-15-keto-prostaglandin F2α. 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.

Direct expression of pig testicular (20β)-hydroxysteroid dehydrogenase in Escherichia coli

Abstract The cDNA coding for pig testicular 3α β (20β)-hydroxysteroid dehydrogenase was expressed in Escherichia coli by placing it under the control of an isopropylthiogalactoside (IPTG) inducible tac promoter. Production of 3α β (20β)-HSD was demonstrated by Western blotting and by catalytic activity with 5α-dihydrotestosterone as a substrate for 3α β - HSD , and progesterone and 17α-hydroxyprogesterone as substrates for 20β-HSD in the presence of NADPH. The 3α β (20β)-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 3α β (20β)-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 3α β (20β)-HSD was not detected suggesting that the N-terminal amino acid was blocked.

3α-Hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20α-hydroxysteroid dehydrogenase ☆

Abstract In earlier studies, two distinct molecules, 20α-HSD-I and 20α-HSD-II, responsible for 20α-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 20α-HSD-I, which mainly catalyzes the reduction of 17α-hydroxyprogesterone to 17α,20α-dihydroxy-4-pregnen-3-one, catalyzes 3α-hydroxysteroid oxidoreductase activity for 5α (or 5β)-androstanes (C19), 5α (or 5β)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5α (or 5β)-androstane substrates rather than 5α (or 5β)-pregnane substrates, and the 5β-isomers rather than 5α-isomers, respectively. Kinetic constants in the reduction for 5α-androstanedione (Km; 3.3 μM, Vmax; 69.7 nmol/min/mg) and 5β-androstanedione (Km; 7.7 μM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17α-hydroxyprogesterone (Km; 26.2 μM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20α-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3α-hydroxy-5α-androstan-17-one were 1.7 μM and 43.2 nmol/min/mg, and 1.2 μM and 32.1 nmol/min/mg for 3α-hydroxy-5β-androstan-17-one, respectively. 20α-HSD activity in the reduction of 17α-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5α-DHT with a Ki value of 2.0 μM. Furthermore, 17α-hydroxyprogesterone inhibited competitively 3α-HSD activity with a Ki value of 150 μM.

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.