Enrico Forchielli

In vitro enzymatic cleavage of the cholesterol side chain in rat testis preparations

Abstract The cholesterol side chain cleavage enzyme in rat testis has been localized in the mitochondrial fraction of the testis homogenate. The enzyme system has an absolute requirement for a pyridine nucleotide, TPNH being the most effective with DPNH, and TPN also effective but to a lesser degree. Cleavage enzyme activity can be enhanced by repeated washing of the mitochondrial fraction with 0.25 M sucrose, which procedure presumably removes inhibiting endogenous cholesterol. This is supported by the observation that this enzyme system is extremely sensitive to the concentration of exogenously added unlabelled cholesterol. EPNH and DPN Inhibit the effectiveness of TPNH probably by inhibiting the enzymatic oxidation of pregnenolone to progesterone and the accumulated pregnenolone then inhibiting the cleavage of its immediate precursor, 20α,22α-dihydroxycholesterol. The DPN also inhibits perhaps by first being converted to DPNH through enzymatic transhydrogenation with TPMH.

In vitro effect of gonadotrophins on the soluble cholesterol side-chain cleaving enzyme system of bovine corpus luteum

Abstract A soluble cholesterol side-chain cleaving enzyme system has been prepared from bovine corpus luteum which can be stimulated in vitro by added gonadotrophins. The gonadotrophins appear to exert their effect on the 20α-hydroxylation of cholesterol.

The δ5-3β-hxirqxysteroid dehydrogenases of corpus luteum and adrenal. II. interaction of c19 and C21 substrates and products ☆

Abstract Incubation of a variety of C 21 -Δ 5 -3β-hydroxy steroids with adrenal and corpus luteum acetone powders in the presence of DBA (3β-hydroxyandrost-5-ene-17-one) resulted in a marked inhibition in the rate of oxidation of the C 21 substrates and preferential oxidation of DHA. DHA-SO 4 was without effect. Androstenedione (androst-4-ene-3,17-dione) was also found not only to inhibit the oxidation of the C 21 -Δ 5 -3β-hydroxysteroids more effectively than DHA, but also inhibited the oxidation of DHA itself. Other Δ 4 -3-ketosterolds such as testosterone (17β-hydroxyandrost-4-ene-17-one) and 11β-hydroxyandrostenedione (11β--hydroxyandrost-4-ene-3, 17-dione) were also found to be effective inhibitors. The inhibition by androstenedione of the C 21 -Δ 5 -3β-hydroxysteroid substrates could not be overcome by increasing the concentration of substrate whereas inhibition of DHA oxidation by androstenedione was clearly competitive for the inhibition could be overcome by increasing the substrate concentration. Progesterone (pregn-4-ene-3,20-dione) was found to be a less potent inhibitor of Δ 5 -3β-ol oxidation, but differed fron androstenedione in that it inhibited the Δ 5 → Δ 4 isomerization step while androstenedione did not. The observed pattern of inhibition and the similarity in relative rates between different tissue preparations suggested the concept of a single enzyme system interacting with the C 21 -Δ 5 -3β-ol substrates whereas the oxidation of the saturated 3β--ol and the Δ 5 -3β-ol, at least for the C 19 substrates, seemed to be mediated by different enzyme systems. Implications of these observations are discussed relative to alteration of steroid biosynthesis in pathologic states of androgen excess and possible roles for the steroid sulfokinases and sulfatases of steroidogenic tissues in the regulation of steroid hormone biosynthesis.

Influence of gonadotrophins on the cholesterol-sidechain cleavage reaction by rat-testis mitochondrial preparations

Abstract 1. 1. Conversion of [26- 14 C]cholesterol to pregnenolone and isocaproic acid by a washed rat-testis mitochondrial preparation proceeded in the presence of certain tricarboxylic acid-cycle intermediates such as succinate, fumarate or isocitrate. TPNH or a TPNH-generating system could be substituted for the tricarboxylic acid intermediates. 2. 2. A significant decrease in the cholesterol sidechain cleavage activity is observed 15 min after hypophysectomy. The decrease in the cleavage activity can be prevented by instituting immediate treatment of the hypophysectomized animal with human chorionic gonadotrophin. 3. 3. Neither ACTH, TSH nor prolactin could replace gonadotrophin in enhancing the cleavage reaction of the hypophysectomized animal. 4. 4. An inverse relationship was observed between rate of cholesterol sidechain cleavage and labeled amino acid incorporation into mitochondrial protein after gonadotrophin treatment.

Δ5 -3-Keto isomerases — specificity and inhibition studies in bovine adrenal homogenate fractions and distribution in rat tissues ☆

Abstract Enzyme(s) catalyzing the conversion of androst-5-ene-3,17-dione (I) to the corresponding Δ4 -3-ketone have been found in a number of rat tissues with the highest specific activity residing in the adrenal. Bovine adrenal homogenates demonstrated Δ5 -3-keto isomerase activity in the soluble, mitochondrial and microsomal fractions with the greatest enzyme concentration in the particulate fractions. Acetone powders prepared from the three bovine adrenal fractions effected the isomerization of I, of pregn-5-ene-3,20-dione (II) and of 17α-methyl-17β-hydroxyandrost-5-en-3-one (III) while cholest-5-en-3-one was not affected, in each fraction the isomerization of I and of II was shown to be catalyzed by separate enzymes although I and III were substrates for the same enzyme. Progesterone markedly inhibited the isomerization of II and to a lesser extent, the isomerization of I. There appears to be no coenzyme requirement for isomerase activity.

Requirements of the cholesterol side-chain-cleaving enzyme system of rat-testis mitochondria

Abstract A 0.25 M sucrose suspension of a washed rat-testis mitochondrial preparation containing the cholesterol side-chain-cleaving enzyme system was incubated in the presence of TPNH and cyanide 1 . It was most active when buffered with 0.02 M Tris-HCl buffer at pH 7.4 as compared with an 0.154 M KCl suspension buffered with 0.066 M phosphate buffer also at pH 7.4. Addition of either Mg 2+ or Ca 2+ resulted in a 7–8-fold increase in rate. In the presence of optimal levels of Ca 2+ (10 μmole), addition of 120 μmoles of KCl resulted in a further 30% increase in the rate of side-chain cleavage. Pre-treatment with gonadotropin of immature and mature rats resulted in a 6-fold increase in the rate in vitro of cholesterol side-chain cleavage by the isolated mitochondrial pellets. However, no effect was observed by gonadotropins added in vitro .

The Δ5-3β-hydroxysteroid dehydrogenase of bovine corpus luteum and adrenal ☆: I. Properties, substrate specificity and co-factor requirements

Abstract The enzymatic conversion of nine Δ 5 -3 β -hydroxysteroids to their corresponding Δ 4 -3-ketosteroids has been studied quantitatively in homogenates and acetone powder preparations of bovine corpus luteum and adrenal cortex. At low substrate concentrations there was little difference in the conversion rates of the seven compounds which reacted. At substrate levels approaching enzyme saturation the relative rates were: androst-5-en-3β-ol-17-one 100, pregn-5-en-3β-ol-20-one 60, pregn- -5-ene-3β,17α-diol-20-one 35, pregn-5-ene-3β,21-diol-20-one 105, pregn- -5-ene-3β,17α,21-triol-20-one 115, pregn-5-ene-3β,11β,17α,21-tetrol-20- -one 160, androst-5-ene-3β,17β-diol 60, cholesterol 0 and pregn-5-en- -3β-ol 0. The same enzymatic potential was observed in both tissues. DPN was the preferred co-factor with conversion rates falling by 50–60 percent when TPN was substituted for DPN. DPNH significantly inhibited the Δ 5 -3 β -ol oxidation and this inhibition could be overcome by addition of excess DPN.

The effect of electron-withdrawing and electron-releasing substituents on the enzymic reduction of steroids

Abstract The Δ 4 -hydrogenases of male rat-liver supernatant fractions convert unsubstitued testosterone derivatives to the 4,5-dihydro-3-keto compounds which are then reduced further to 3-hydroxysteroids by saturated 3-keto reductases which are also present in these fractions. Introduction of a halogen substituent at the C-2α, C-4, C-6α or C-6β position not only increases the reduction rate but promotes an abnormal enzymic pathway, Δ 4 -3-ol formation. This reduction, which proceeds in the presence of TPNH or DPNH is believed to be mediated by the saturated 3-keto reductases and is rationalized on the basis of electronic destabilization of the α,β-unsaturated ketone moiety by electronegative substituents with stabilization of the proposed transition state for reduction. In contrast, methyl substitution retards reduction which is explained on electronic and in some cases steric grounds.

A Comparison of Infrared Frequencies of 19-Nor Steroids and their 19-Methyl Analogues

The infrared absorption spectra of thirteen 19-nor steroids in the C-19 series were recorded by the potassium bromide dispersal technique It was found that a shift to higher frequencies occurred for the bands that have been correlated with structures in saturated 3-hydroxysteroids, 3α, 5α shifted from 998 to 1017 cm−1, 3β, 5α from 1042 to 1060 cm−1, 3α, 5β from 1035 to 1060 cm−1, and 3β 5β from 1033 to 1055 cm−1. A band near 881 cm−1 helped identify Δ4-19-nor structures, while a similar band near 885 and/or 825 cm−1 seemed characteristic for the Δ1-19-nor compound

Androgen levels in ovarian vein plasma and in vitro biosynthesis of androgens by ovarian tissue obtained from females with polycystic ovarian disease

Ovarian vein plasma samples obtained from 4 hirsute and/or virilized subjects were analyzed for their androgen and estrogen content. In addition, ovarian wedges obtained at the same time from 2 of the 4 patients were incubated in vitro with a variety of precursors and the conversion to testosterone, androstenedione, and epitestosterone was assessed.