Tamaoki Bun-Ichi

Steroidogenesis and cell structure: Biochemical pursuit of sites of steroid biosynthesis

Abstract Steroidogenesis is discussed at the different levels of the whole body, organ, cell, subcellular components and biomembrane, in order to pursue the sites of production of steroid hormones in the body. From the information on the sites of steroid transformations in a cell, intracellular transport of steroidal precursors, intermediates, and final products are integrated together with the biosynthetic pathways of the steroid hormone.

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.

Testosterone metabolism in rooster comb

Abstract After testosterone was incubated with the cell-free homogenates of rooster comb in air in the presence of NADPH, 5β-androstane-3β,17β-diol was identified as the major metabolite and 5α-dihydrotestosterone, 5β-dihydrotestosterone, 5α-an-drostane-3α, 17β-diol and 5β-androstane-3α,17β-diol were also obtained. Among the subcellular fractions, 5α-reductase activity was mostly detected in the microsomal fraction (10000–105000 × g ), while 5β-reductase activity was exclusively found in the cytosol fraction (supernatant fluid at 264000 × g ). 3α-Hydroxysteroid dehydrogenase (EC 1.1.1.50) which prefered 5α-dihydrotestosterone as the substrate was localized in the cytosol and microsomal fractions, but the dehydrogenase which prefered 5β-dihydrotestosterone was found in the microsomal and mitochondrial (800–6000 × g ) fractions. 3β-Hydroxysteroid dehydrogenase (EC 1.1.1.51) which reduced 5β-dihydrotestosterone but not its 5α-epimer was found mainly in the cytosol fraction. The 3α- and 3β-hydroxysteroid dehydrogenases in the cytosol and microsomal fractions of the comb utilized specifically 4-pro-S-H of NADPH for reduction of the oxo group of 5α- and 5β-dihydrotestosterones. With aging, 5α-reductase activity showed a decrease, whereas 5β-reductase remained unchanged. Among the metabolites, the 5α-hydrogenated steroids showed significant androgenic activities, but the 5β-hydrogenated steroids showed no comparable activities upon cockerel comb. Androgenic activity of 5α-dihydrotestosterone upon the comb was significantly reduced by simultaneous administration of 5β-dihydrotestosterone.

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.

Fate of molecular oxygen required by endocrine enzymes for the side-chain cleavage of cholesterol

Abstract In order to study the role of molecular oxygen in the course of pregnenolone synthesis from cholesterol, four substrates such as cholesterol, 20α-hydroxycholesterol, 22-hydroxycholesterol and 20α,22 R -dihydroxycholesterol were separately incubated with rat adrenal mitochondrial preparations in an 18 O 2 -enriched atmosphere. The progesterone fractions which were derived from cholesterol and 22-hydroxycholesterol were found to contain an atom of 18 O per steroid molecule as the 20-oxo group, while 20α-hydroxycholesterol and 20α,22 R -dihydroxycholesterol were converted into progesterone which was devoid of 18 O. These results indicated that introduction of an oxygen atom from molecular oxygen at the 20α-position of cholesterol was essential for the side-chain cleavage, as the oxygen atom, in the form of the 20-oxo group of pregnenolone and also progesterone, originated from the oxygen of the hydroxy group introduced in the 20α-position of cholesterol prior to the cleavage.

On the role of the cytosol receptors in the incorporation of androgens into the prostatic nuclei of rat

Abstract By continuous sucrose density gradient centrifugation, two kinds of macromolecules or receptors which bound to androgens were detected in the cytosol fraction of rat ventral prostates, and their sedimentation coefficients were determined as 9 S and 5 S. When the competitive bindings to the receptors were examined between testosterone and dihydrotestosterone, and between dihydrotestosterone and androstanediol, the receptors showed the highest affinity to dihydrotestosterone among them. Several anti-androgenic reagents, such as cyproterone, its 17α-acetate, SK&F 7690 and estradiol-17β suppressed the bindings of dihydrotestosterone and testosterone to the cytosol receptors. By these anti-androgens, the binding of testosterone to the receptors was more severely reduced than that of dihydrotestosterone. Radioactive testosterone was previously incubated with the cytosol fraction or its subfractions and then the incubation mixtures were incubated with the nuclei in the presence of NADPH. Then, it was found that the nuclei selectively retained dihydrotestosterone transformed from testosterone. The highest retention of radioactivity by the nuclei was caused by addition of the cytosol subfraction precipitated by 40% saturation of ammonium sulphate. Since this subfraction consisted mostly of the 9 S receptor, it is suggested that the 9 S receptor is directly involved in the transport of dihydrotestosterone into the nuclei. Time course study of the incorporation of the androgens to the nuclei indicated that dihydrotestosterone was incorporated into the nuclei more rapidly and efficiently than testosterone, in the presence of the 9 S receptor.

In vitro metabolism of testosterone in seminal vesicles of rats

Abstract When [4-14C]-testosterone was incubated in vitro with cell-free homogenates (800 g supernatant fluid) of rat seminal vesicles. 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol were identified as the metabolites. Both the nuclear and microsomal fractions most efficiently converted testosterone to the above 5α-hydrogenated metabolites. When testosterone was pre-incubated with the cytosol fraction (105,000 g supernatant fluid), consequent 5α-hydrogenation by the nuclear and microsomal fractions was decreased. If the cytosol fraction was heated at 100°C, the inhibitory effect upon the enzyme activity diminished. 3α-Hydroxysteroid dehydrogenase activity upon 5α-dihydrotestosterone was detected almost exclusively in the cytosol fraction among the subcellular fractions. NADPH was found as the most preferable cofactor for Δ4-5α-hydrogenase and 3α-hydroxysteroid dehydrogenase. Δ4-5α-Hydrogenase and 3α-hydroxysteroid dehydrogenase reduced testosterone and 5α-dihydrotestosterone respectively by the transfer of 4-pro-S-hydrogen of NADPH. Optimal pH ofΔ4-5α-hydrogenase in the nuclear and microsomal fractions was from 5.7 to 5.9 and that of the 3α-hydroxysteroid dehydrogenase in the cytosol fraction was about 6.3. Optimal temperature was around 37°C for Δ4-5α-hydrogenase in the two subcellular fractions, and 50°C for 3α-hydroxysteroid dehydrogenase. Cu2+, Cd2+, Hg2+, Zn2+ and p-chloromercuribenzoate markedly inhibited both Δ4-5α-hydrogenase and 3α-hydroxysteroid dehydrogenase activities, whereas Co2+ selectively inhibited Δ4-5α-hydrogenase activity. Δ4-5α-Hydrogenase activity was reduced by EDTA and o-phenanthroline.