Binding Of Pregnenolone Research Articles

Conformational selection dominates binding of steroids to human cytochrome P450 17A1

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

The influence of Sutherlandia frutescens on adrenal steroidogenic cytochrome P450 enzymes

Aim of the study The aim of this study was to investigate whether Sutherlandia frutescens, subsp. microphylla (family: Fabaceae/Leguminosa), which is traditionally used to treat symptoms of chronic stress generally associated with increased circulating glucocorticoids, influences the biosynthesis of these glucocorticoids. Methods We investigated the interaction of Sutherlandia frutescens with cytochrome P450 enzymes, CYP17 and CYP21, which catalyse key reactions in glucocorticoid biosynthesis. The binding of progesterone and pregnenolone to these enzymes and their metabolism were assayed in the presence of extracts and the bioactive compounds, l-canavanine, pinitol, GABA, flavonoids and triterpenoid glucosides present in the shrub. Results While the aqueous and methanol extracts inhibited the type I progesterone-induced difference spectrum ( p < 0.05), inhibition of pregnenolone binding ( p = 0.25) was negligible, with the aqueous extract exhibiting greater inhibition. The triterpenoid fraction inhibited both the type I pregnenolone- and progesterone-induced difference spectra and elicited a type II difference spectrum in the absence of substrate. Both pregnenolone and progesterone metabolism were inhibited by the aqueous extract, the inhibition of CYP21 being greater than that of CYP17, influencing the flux through glucocorticoid precursor pathways. Conclusion This attenuation of adrenal P450 enzymes may thus demonstrate a possible mechanism by which Sutherlandia frutescens reduces glucocorticoid levels and alleviates symptoms associated with stress.

The Effect of Sutherlandia frutescens on Steroidogenesis: Confirming Indigenous Wisdom

Sutherlandia frutescens (Cancer bush), a Southern African indigenous plant, is traditionally used to treat stress related maladies linked to the endocrine system. Extracts of the shrub were used to investigate the claimed stress‐relieving properties of the shrub. Dysregulation of the stress response is associated with elevated glucocorticoid levels. A model of chronic intermittent immobilization stress was investigated in 40 adult male Wistar rats to determine the effect of Sutherlandia. Immobilization stress resulted in increased corticosterone levels in the control group while rats receiving Sutherlandia extract showed significantly decreased corticosterone levels (P < 0.005). Since the biosynthesis of glucocorticoids in the adrenals is catalyzed by the cytochrome P450‐dependent enzymes, the influence of Sutherlandia extracts on adrenal steroidogenesis was determined in ovine adrenocortical microsomes and mitochondria, using spectral binding and enzyme conversion assays. Water extracts showed inhibition of substrate binding to cytochrome P450 21‐hydroxylase (CYP21) by 38% and cytochrome P450 11β‐hydroxylase (CYP11B1) by 60%. The conversion of progesterone and pregnenolone was inhibited by 34% and 30%, respectively. Subsequent extractions with chloroform and methanol showed inhibition of substrate binding and conversion with hydrophobic compounds exhibiting a greater inhibitory effect on deoxycorticosterone binding to CYP11B1 (30%) and on progesterone binding to CYP21 (50%). The inhibition of binding of pregnenolone to CYP17 by the chloroform extract was 62%, with negligible inhibition by the methanol extract. The chloroform extract showed a greater inhibitory effect than the methanol extract on progesterone and pregnenolone metabolism (20%–50%).

MAPREG: toward a novel approach of neuroprotection and treatment of Alzheimer's disease.

MAPREG (microtubule-associated protein/neurosteroidal pregnenolone) is a start-up company that was created in October 2000. Its acronym recalls the basic discovery (Murakami et al., 2000) from which drug(s) will hopefully be developed that are useful for neuroprotection and repair in conditions such as post-traumatic and postischemic lesions, as well as defects proper to normal aging and neurodegenerative diseases, that is, principally Alzheimer's disease. Pregnenolone, the main steroid synthesized from cholesterol in the nervous system (therefore, a neurosteroid), binds specifically with high affinity (> or = 40 nM) to microtubule-associated protein 2 (MAP2), a protein family involved in the assembly and stabilization of microtubules made from tubulin alpha and beta polymers, and in the bundling of several microtubules by MAP2 projection arms. Pregnenolone binding increases MAP2-induced microtubule polymerization, when purified tubulin and MAP2 are coincubated in GTP containing buffer at 37 degrees C. Therefore, MAP2 can be considered as a receptor for a novel mechanism of steroid action. The underlying principle and its potential pharmacological consequences are described in an INSERM patent (FR 0003430; March 17, 2000). MAPREG has established its own laboratory in a space rented to Bicêtre hospital, near the research building of INSERM, where two of the main founders of the company (Drs. E. Baulieu and P. Robel) work. The company has been quite successful, largely thanks to the support of ISOA (attributed in October 2002). A lead compound (pregnenolone derivative) was tested and patented by MAPREG early in 2003 (FR 0300507; January 17, 2003). Activities and results reported at the ISOA meeting on Oct. 2, 2003, include in vitro basic studies, in vitro and in vivo neuroprotection trials in rodent systems, and studies with human cells and an AD transgenic mouse model.

Crystal Structure of Human Cholesterol Sulfotransferase (SULT2B1b) in the Presence of Pregnenolone and 3′-Phosphoadenosine 5′-Phosphate: RATIONALE FOR SPECIFICITY DIFFERENCES BETWEEN PROTOTYPICAL SULT2A1 AND THE SULT2B1 ISOFORMS

The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket.

Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly.

Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30-50 nM range. The best competitors (in decreasing order) are pregnenolone sulfate, progesterone, Delta5-pregnene-3beta,20alpha-diol, and 3beta-hydroxy-5alpha-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40-60 kDa, enriched in [(3)H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [(3)H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2-tubulin interaction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging.

Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.

Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07–1.09 g/cm 3). Preincubation with digitonin perturbed the buoyant density of this fraction (to1.10–1.14 g/cm 3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F 2α) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5–15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 μM. In the presence of digitonin, [ 3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [ 3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [ 3H]progesterone (half-maximal displacement of specific binding (IC 50) at about 60 nM) whereas androgens were less potent (IC 50, 500–3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were neffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3,4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.

17-Hydroxylase and andien-β synthetase activities in immature pig testis microsomal fraction: Kinetic studies of the pregnenolone binding site and possible intermediates of the reactions

The microsomal fraction from the testes of immature pigs can convert pregnenolone to 17-hydroxypregnenolone and also to 5,16-androstadien-3β-ol (andien-β). The available evidence supports the hypothesis that both these reactions are catalysed by one enzyme, cytochrome- P450 17 α . In the absence of cytochrome b 5, 17-hydroxypregnenolone will be the major product but that if cytochrome b 5 is present in sufficient quantity, andien-β becomes a major product. The point of divergence between the conversion of pregnenolone to either 17-hydroxypregnenolone or andien-β was investigated using enzyme kinetic analysis to determine whether 16α-hydroxypregnenolone, 20β-hydroxypregnenolone or 16-dehydropregnenolone could be specific intermediates to one reaction or the other. Product inhibition by 17-hydroxypregnenolone and andien-β was competitive for both 17-hydroxylase and “andien-β synthetase” supporting the current view of a common active site for both reactions. 16α-Hydroxypregnenolone was a very poor competitive inhibitor of 17-hydroxylase and andien-β synthetase with K i(app) values many fold greater than the K m(app) for pregnenolone or the K i(app) for reaction product, rendering it unlikely that 16α hydroxylation is a key intermediary step in either pathway. 20β-Hydroxypregnenolone was a more potent inhibitor of andien-β synthetase than of 17-hydroxylase and for the latter enzyme activity, the K i(app) was lower than that for 17-hydroxypregnenolone itself. However, for andien-β synthetase, 20β-hydroxypregnenolone may be an early intermediate as the K i(app) was consistent with the affinity for the active site being intermediate between the K m(app) for andien-β. 16-Dehydropregnenolone was equipotent at inhibiting 17-hydroxylase and andien-β synthetase activities suggesting that 16-dehydropregnenes may be involved in the stages immediately prior to C21 side-chain cleavage.

Immunocytochemical localization of the 34 KD pregnenolone-binding protein to fasciculata and reticularis cells and a novel 32 KD protein specific for reticularis cells in guinea pig adrenal cortex.

Two proteins were isolated and purified from guinea pig adrenal cortex: a 34 KD protein that specifically binds pregnenolone (product of the rate-limiting step in steroidogenesis), and a novel co-purifying 32 KD protein that has not been characterized. Specific antisera were generated and used for immunocytochemical analysis. The 34 KD and 32 KD proteins were specific for the adrenal cortex and were absent from other tissues, including the testis. The 34 KD pregnenolone binding protein (PBP) was localized to zona fasciculata and zona reticularis cells and absent from zona glomerulosa cells. Thus, the PBP was absolutely correlated with ACTH-regulated steroidogenic cells, whereas steroidogenic cells regulated by other peptide hormones did not contain the PBP. This finding suggests a functional relationship between the PBP and ACTH. A second interesting finding was that a novel 32 KD co-purifying protein localized to the zona reticularis and was absent from the zona glomerulosa and the zona fasciculata. The 32 KD protein can therefore serve as an excellent marker for the reticularis cell of the adrenal cortex.

The Nuclear Conversion of Pregnenolone to Progesterone and Subsequent Binding to the Nuclear Progesterone-Binding Protein in the Guinea Pig Adrenal Cortex: A Possible Regulatory Role for the Pregnenolone-Binding Protein

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.

Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.

Isoelectric focusing of the Mr 34,000 pregnenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroid-binding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3H]pregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high- and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 proteins with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms of Mr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was approximately 10-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolone-binding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule.

Pregnenolone binding sites in the rat olfactory bulb

High concentrations of pregnenolone and its sulfate have been found in several areas of rat and human brain and seem to be controlled by local mechanisms. In the present experiments we have demonstrated pregnenolone binding sites in the cytosolic fraction of the rat olfactory bulb. The pregnenolone binding component showed a K d = 2.34 ± 0.66 × 10 −7 M and N max = 7.25 ± 1.20 pmol/mg protein. Pregnenolone, pregnenolone sulfate and 17OH-pregnenolone competed equally for the binding sites while other steroids were less competitive. Protease and trypsin inhibited binding by 48 and 60% respectively. Sucrose density gradient analysis showed a minor peak at 4.6 s and a major one at 3.6 s. After gel filtration chromatography the pregnenolone binding component appeared as 2 peaks corresponding to molecular weights of approximately 150 and 220 kDa. Heating at 60°C increased binding by 150%. These results indicate that the olfactory bulb pregnenolone binding component is complex in nature. Rat plasma also bound pregnenolone. Plasma binding sites could be partially differentiated from those in the olfactory bulb on the basis of susceptibility to lipoprotein lipase, effect of heating and mobility during polyacrylamide gel electrophoresis.

Brain mitochondrial cytochrome P-450scc: Spectral and catalytic properties

The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity. The system has been found to bind cholesterol and 11-deoxycorticosterone, producing type I difference spectra, whereas the binding of pregnenolone induced a reverse type I difference spectrum. Inhibitors of cytochrome P-450-linked monooxygenase activities produced type II spectra. The formation of labeled pregnenolone after incubation of brain mitochondria with [4- 14C]cholesterol has been obtained, and this formation was inhibited by glutethimide, a specific inhibitor of cytochrome P-450scc. The functional significance of this enzymatic activity is discussed.

A comparative study of estramustine and pregnenolone binding to prostatic binding protein: Evidence for subunit cooperativity

The binding of estramustine, a nitrogen mustard derivative of oestradiol to purified rat prostatic binding protein was studied as a test for a possible identity between this protein and the very similar estramustine-binding protein, described by Forsgren et al. [5]. In accordance with this hypothesis estramustine binds to purified prostatic binding protein with a high affinity (2.5 × 10 7M −1). This affinity markedly exceeds the affinity of pregnenolone for this protein (0.9 × 10 6M −1) or for a complex of prostatic binding protein, with prostatic proline-rich polypeptide, (4.7 × 10 6M −1). In competition experiments estramustine completely suppresses the binding of [ 3H]pregnenolone, whereas the binding of [ 3H]estramustine is only partially suppressed by pregnenolone, even at high concentrations. Prostatic binding protein was separated in its F- and S-subunit by DEAE-Sepharose chromatography performed in the presence of 8 M urea. Only the S-subunit, most probably in its dimer form. displays marked estramustine and pregnenolone binding, with affinities of respectively 3.7 and 1.2 × 10 6M −1. Recombination of both subunits results in a strong increase of estramustine binding, but not of pregnenolone binding.

114. A comparative study of pregnenolone and estramustine binding to the prostatic binding protein of rat ventral prostate

114. A comparative study of pregnenolone and estramustine binding to the prostatic binding protein of rat ventral prostate

Pregnenolone efflux from mitochondria of bovine adrenal cortex.

Pregnenolone efflux from bovine adrenal cortex mitochondria was measured with the aid of rapid separation of the organelles from the incubation by centrifugation through silicon oil. In the absence of bovine serum albumin or cytosolic supernatant (100 000 x g), an equilibrium distribution of newly synthesised pregnenolone was attained after 2 min at 30 degrees C; 20% of total pregnenolone was located outside the mitochondria. The presence of 13 mg albumin/ml or 13 mg supernatant protein/ml increased the exogenous proportion of pregnenolone to over 90% and 75% respectively without affecting the rate of cholesterol side-chain cleavage. The binding of pregnenolone by both albumin and supernatant was non-specific and non-saturable. It is suggested that in vivo the mitochondrial membranes do not present a significant barrier to pregnenolone efflux from the organelle.

Effect of oestrogen treatment on testicular LH/HCG receptors and endogenous steroids in prostatic cancer patients.

LH/hCG binding and concentrations of pregnenolone, progesterone, 17-hydroxy-progesterone, androstenedione, testosterone and 5 alpha-dihydrotestosterone were measured in testicular tissue obtained from patients undergoing orchiectomy for prostatic cancer. One group of the orchiectomized patients had received an injection of 80 mg polyestradiol phosphate 1-9 days before the operation, another group were treated with monthly injections of the oestrogen for several months, and the remainder were not treated with oestrogen. In non-oestrogen treated patients (n = 8) the hCG-binding capacity was 28.0 +/- 15.0 (SD) ng/g and the equilibrium association constant of binding was 0.69 +/- 0.38 x 10(10) M-1. The binding capacity was significantly (P less than 0.025) lower (10.3 +/- 8.7 ng/g) in patients (n = 6) receiving the first oestrogen injection 3-9 days prior to the operation. A further decline to the level of 1.77 +/- 1.03 ng/g occurred in patients (n = 3) treated over 3 months with the oestrogen injections. At the same time, no significant changes were observed in peripheral serum LH and FSH levels. In the testicular endogenous steroid levels, statistically significant decreases were seen 9 days after the oestrogen injection in pregnenolone (from 505 +/- 315 ng/g to 138 +/- 86 ng/g) and in testosterone (from 669 +/- 243 ng/g to 254 +/- 49 ng/g) whereas no significant changes could be seen in the levels of the other steroids analysed. This study gives further evidence for the direct inhibitory action of oestrogens on human testicular steroidogenesis and suggests that the loss of testicular luteinizing hormone receptors may be one facet of this inhibitory action.

Molecular nature of interaction of steroids with biomembranes related to androgen biosynthesis

Pregnenolone and progesterone were found to be differentially concentrated in the membrane fractions from Leydig cell tumors and cryptorchid testes when equilibrium between membranes and the steroid containing buffer was achieved without metabolism. On the other hand, androstenedione was not significantly bound to any of the membranes. This concentrating system for pregnenolone and progesterone had high capacity and a low association constant. The amounts of pregnenolone bound to the mitochondria-rich fraction (MRF), smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) were 4.8, 9.9 and 7.3 nmol/mg protein, respectively, when these fractions were incubated with 5 nmol of pregnenolone in 5 ml of the buffer. These differences, however, almost disappeared when the binding data were viewed in relation to the amount of membraneous phospholipid; that is, 0.3 for MRF, 0.29 for SER and 0.34 nmol/μg Pi of phospholipid for RER. The binding of pregnenolone to 3β-ol-dehydrogenase would be negligible, since tumor membranes which showed considerable variation in this enzymatic activity had a fairly constant capacity of the pregnenolone binding. Progesterone is bound to these fractions in a similar manner to pregnenolone. These results might suggest that the steroid-bio-membrane interaction occurs almost exclusively with phospholipids in membranes. The partition coefficients between the phospholipid in SER and the aqueous suspending medium were calculated to be 1.5 × 10 3 for pregnenolone, 2 × 10 2 for progesterone and 4.5 for androstenedione.

Pregnenolone-Binding Proteins in the Rat Adrenal Cortex

Rat adrenal cortex contains a protein(s) that binds pregnenolone with high affinity. This binding, demonstrated by gel filtration and by a dextran-coated charcoal method, was associated with the cytosol and with fractions solubilized by sonication from the mitochondria and microsomes. The binding of pregnenolone was saturable and was inhibited by mercurials and proteolytic enzymes. Pregnenolone-binding was not influenced by the presence of progesterone, deoxycorticosterone, corticosterone or aldosterone, but was inhibited by steroids with a 3beta-hydroxy-5-ene structure similar to pregnenolone, and by hydroxymethylene steroid and cyanoketone. We suggest that this protein is involved in the intracellular transport and retention of pregnenolone within adrenal cortical cells.