Inano Hiroshi

The influence of various factors upon testicular enzymes related to steroidogenesis

Abstract The 17α-hydroxylation of pregn-4-ene-3,20-dione by rat testicular microsomal fraction was inhibited by the addition of 17α,20β-dihydroxypregn-4-en-3-one, 20β-hydroxypregn -4-en-3-one and chemical inhibitors such as ethylenediaminetetraacetic acid and p -chloromercuribenzoate to the medium. The side-chain cleavage of 17α-hydroxypregn-4-ene-3,17-dione by the rat testicular microsomal fraction was inhibited by 17α,20α-dihydroxy-, 17α,20β-dihydroxy-, 20α-hydroxy-, and 20β-hydroxypregn -4-en-3-one, 21-hydroxypregn-4-ene-3,20-dione and p -chloromercuribenzoate. The 17β-reduction of androst-4-ene-3,17-dione by the rat testicular microsomal fraction was inhibited by p -chloromercuribenzoate, but not by iodoacetamide or NaCN. The 20α-hydroxysteroid dehydrogenase activity was inhibited by p -chloromercuribenzoate, but activated by the addition of ethylenediaminetetraacetic acid. The reduction of the 20-keto group of 17α-hydroxypregn-4-ene-3,20-dione was significantly enhanced under anaerobic conditions even in the presence of testicular microsomal fraction, suggesting that molecular oxygen was the decisive factor in directing the two enzymic reactions: the side-chain cleavage and the 20-keto reduction of 17αhydroxypregn -4-ene-3,20-dione. Moreover, the activity of the lyase for the side-chain cleavage of 17α-hydroxypregn-4-ene-3,20-dione was competitively inhibited by 17α, 20α-dihydroxypregn-4-en-3-one which was produced from 17α-hydroxypregn-4-ene3,20 -dione by the 20α-hydroxysteroid dehydrogenase. These findings would suggest some regulatory role for the 20α-hydroxysteroid dehydrogenase, and the products of its action, upon androgen synthesis.

Testicular and adrenal 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

Abstract The purified multifunctional enzyme, 3β-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3β-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.

Studies on enzyme reactions related to steroid biosynthesis: II. submicrosomal distribution of the enzymes related to androgen production from pregnenolone and of the cytochrome P-450 in testicular gland of rat

Abstract A testicular microsomal fraction containing the enzyme systems necessary for the production of testosterone from pregnenolone was divided in two subfractions by sucrose density gradient centrifugation in the presence of CsCl. When the subfractions were examined by electron microscope, one consisted mainly of smooth-surfaced particles, the other of rough-surfaced particles with ribosomes on their outer surface. Enzymic and spectrometric investigation of these subfractions revealed that most of the enzyme activities related to the androgen synthesis, which remained after the gradient centrifugation, and also of the cytochrome P-450, which would be a direct site of activation of molecular oxygen for the 17α-hydroxylation and the side chain cleavage, were localized in the smooth-surfaced submicrosomal fraction. The enzyme activities of the submicrosomal fraction which were diminished by the gradient centrifugation were largely restored by addition of the heated 105,000 × g supernatant fluid. Enzyme inhibitors such as Amphenone B (3,3-bis ( p -aminophenyl)-2-butanone dihydrochloride) and SKF-525A (2-diethylaminoethyl 2.2-diphenylvalerate hydrochloride) inhibited the testicular microsomal 17α-hydroxylase and the C 17 –C 20 lyase competitively. The apparent inhibition constants of Amphenone B were 9.17 × 10 −5 M against 17 α-hydroxylase and 2.45 × 10 −4 M against C 17 -C 20 lyase, and those of SKF-525 A were 1.61 × 10 14 M against 17α-hydroxylase and 1.28 × 10 −5 M against the C 17 –C 20 lyase.

Localization of the Δ5-3β-hydroxysteroid dehydrogenase and 21-hydroxylase activities in smooth-surfaced microsomes of adrenals

Abstract Rat adrenal microsomes were subfractionated into the smooth and rough-surfaced microsomes by a density gradient centrifugation in the presence of CsCl. Two microsomal enzyme activities related to corticoidogenesis, Δ5-3β-hydroxysteroid dehydrogenase coupled with Δ5-Δ4 isomerase and 21-hydroxylase were predominantly located in the smooth-surfaced microsomes which bore no ribosomes on their outer surface.

Regulation of testosterone biosynthesis in rat testes by 7α-hydroxylated C19-steroids

Abstract 7α-Hydroxyandrostenedione and 7α-hydroxytestosterone inhibited the transformation, in vitro , of pregnenolone to progesterone by the Δ 5 -3β-hydroxysteroid dehydrogenase coupled with the Δ 5 -Δ 4 isomerase in rat testicular microsomal fraction (10000–105000 × g precipitate). The inhibition by the 7α-hydroxylated steroids on the enzyme system was competitive, and the inhibitor constant was estimated to be 56 μM for 7α-hydroxyandrostenedione and 385 μM for 7α-hydroxytestosterone. Substrate constants for the Δ 5 -3β-hydroxysteroid dehydrogenase for pregnenolone were 67 and 64 μM, respectively, in the presence of 7α-hydroxyandrostenedione and 7αhydroxytestosterone. 7α-Hydroxytestosterone inhibited the testicular 7α-hydroxylase activity, but not significantly the 17β-hydroxysteroid dehydrogenase, whereas 7αhydroxyandrostenedione inhibited both enzymic activities. Other testicular enzymic activities related to steroid metabolism, such as the testicular 17α-hydroxylase, C 17 -C 20 -lyase and 20α-hydroxysteroid dehydrogenase were not influenced in vitro by the two 7α-hydroxylated steroids. When 7α-hydroxyandrostenedione was incubated with subcellular fractions of rat testes, no metabolite other than 7α-hydroxytestosterone was obtained. When the testicular microsomal fraction suspended in 0.25 M sucrose solution was stored at 0°, the activity of the 17β-hydroxysteroid dehydrogenase decreased nearly exponentially with the time of storage, but the activity of 7α-hydroxylase remained without decrease up to the 5th day of storage. From the results obtained, the possibility of an intracellular regulation of testosterone production by 7α-hydroxylation and its 7α-hydroxylated steroids is proposed.

Studies on enzyme reations related to steroid biosynthesis—III. Distribution of the testicular enzymes related to androgen production between the seminiferus tubules and interstitial tissue

Abstract In order to examine intercellular distribution of several enzymes related to steroidogenesis in rat testicular tissue, the decapsulated tissue was manually separated into seminiferous tubule and interstitial cell fractions. When 14 C-progesterone was incubated with the two fractions aerobically in the presence of NADPH, 17α-hydroxyprogesterone was obtained as the major product in case of interstitial cell fraction but no significant conversion of the substrate was detected by incubation with seminiferous tubule fraction. When 17α-hydroxyprogesterone was employed as substrate, testosterone was obtained as the main product by incubation with interstitial cell fraction, while in the case of seminiferous tubules, 17α,20α-dihydroxypregn-4-en-3-one was detected as the major metabolite but not C 19 -steroids. From the above results, activities of 17α-hydroxylase and C 17 -C 20 lyase were located in the interstitial tissue, while 20α-hydroxysteroid dehydrogenase was concentrated in the seminiferous tubules.

Chemical modification of tyrosine residues in active-site of human placental estradiol 17β-dehydrogenase by tetranitromethane

Abstract The native estradiol 17β-dehydrogenase purified from human placenta was rapidly inactivated by tetranitromethane, which is a reagent for chemical modification of tyrosine and cysteine residues. The inactivation followed pseudo-first-order reaction kinetics, and the activity was partially recovered by addition of dithiothreitol. On the other hand, the enzyme sulfhydryl-blocked by 5,5′-dithiobis(2-nitrobenzoic acid) was treated with tetranitromethane, and the activity was assayed in the presence of dithiothreitol. Tetranitromethane inactivated the enzyme in a time-dependent manner, following pseudo-first-order kinetics. The rate of inactivation was significantly decreased by addition of NADP(H), 2′-AMP, 5′-ATP, 2′,5′-ADP, 3-pyricline aldehyde-DPN and 3-acetylpyridine-DPN(reduced form). These results suggest that the tyrosyl residues are located at or near the cofactor-bincling site of the estradiol 17β-dehydrogenase and play an essential role in the catalytic function of the enzyme.

Photochemical inactivation of human placent al estradiol 17β-dehydrogenase in the presence of 2,3-butanedione

Abstract Estradiol 17β-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen ( 1 Δ g or 1 ∑ g + states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP + , the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP + was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.

Porcine testicular 17β-hydroxysteroid dehydrogenase: Affinity chromatography with dye-ligand agarose and demonstration of multiple forms of the enzyme

Abstract 17β-Hydroxysteroid dehydrogenase of porcine testes was purified by an affinity chromatography on agarose coupled with Procion Red HE3B. The purified enzyme preparation approached apparent homogeneity checked by SDS-polyacrylamide gel electrophoresis. By zymographic analysis of the dehydrogenase preparation on polyacrylamide gel disc electrophoresis, this preparation was separable into at least three protein bands which had the dehydrogenase activities. Furthermore, by isoelectric focusing in polyacrylamide gel, four enzyme fractions were distinctly separated, and their isoelectric points were determined as pH 4.9, 5.0, 5.5, and 5.6. The heterogeneity of the dehydrogenase was mainly due to the presence of a different net charge on the molecule, but not difference in molecular size. The molecular weight of these distinct enzymes was estimated as 34,000 by the disc gel electrophoresis. According to our zymographic analysis, this enzyme catalyzed oxidation of testosterone to androstenedione in the presence of NADP + but did not significantly convert androst-5-ene-3β,17β-diol to dehydroepiandrosterone.

Active form of 17β-hydroxysteroid dehydrogenase of porcine testes

17β-Hydroxysteroid dehydrogenase (E.C. 1.1.1.64) of porcine testes was centrifuged at 10° over a sucrose density gradient which contained androstenedione and NADPH, and then the gradient was fractionated. The amount of testosterone was quantitated in each fraction immediately after the centrifugation, and also after further incubation of each fraction at 37° for 3 h. From distributions of the testosterone over the gradients, it was concluded that the active form of the dehydrogenase occurs as its monomer with sedimentation coefficient 3.11 s and molecular weight, 35,400 daltons.