High affinity ryanodine binding sites in rat liver endoplasmic reticulum

The binding of [3H]ryanodine to liver microsomal subfractions was investigated. The smooth microsomal membranes were enriched with ryanodine binding sites and also with a polypeptide of 360 kDa. Caffeine completely inhibited [3H]ryanodine binding. Ryanodine also affected the membrane Ca2+ permeability. At low concentrations (less than 10 microM) ryanodine stimulated Ca2+ efflux and at higher concentrations (greater than 50 microM) it blocked Ca2+ efflux. These results suggest that hepatic microsomes contain ryanodine binding sites which can modify the membrane permeability for Ca2+.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate

Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms (Ka = 0.5-2.9 x 10(10) M-1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates.     

Characterization of estrogen-binding sites associated with the endoplasmic reticulum of rat uterus

Microsomes prepared from rat uterine homogenates harbor high-affinity (Ka = 10(10) M-1), low-capacity binding sites for estrogens. Previous work from our laboratory has demonstrated that these estrophiles are located on endoplasmic reticulum and are not cytosolic contaminants of the membrane preparation. Subfractionation of microsomes into granular and agranular membranes and polysomes revealed approximately equal distribution of estrogen-binding activity among each of these constituents. These binding sites were fully extractable with 0.6 M KCl. Microsomal estrophiles solubilized under conditions of low ionic strength and complexed with estradiol migrated as 8S forms on continuous sucrose gradients. In the presence of 0.4 M KCl, the solubilized binding sites exhibit a sedimentation coefficient of 4S. Extracted binding sites do not undergo heat-induced transformation from a 4S to 5S species. The monoclonal antibody JS34/32 interacted with the endoplasmic reticulum-associated estrogen-binding sites when present in 50-fold molar excess, but not at lower antibody to binding site ratios. In comparison, the rat uterine cytosolic estrogen receptor formed complexes with JS34/32 at antibody to receptor ratios as low as 2:1. These results suggest that the endoplasmic reticulum possesses estrogen-binding sites with biochemical properties that differ from those of the classically described cytosolic (loosely associated nuclear) estrogen receptor.     

Optimal radioligand exchange conditions for measurement of occupied androgen receptor sites in rat ventral prostate

This study was undertaken to define optimal conditions for exchange of ³H-R-1881 with endogenous hormone bound to androgen receptor (AR) sites in homogenates of rat ventral prostate (RVP) of mature animals. To minimize inactivation of AR binding sites under exchange conditions, extracellular proteases present in RVP were removed by mincing and washing tissue fragments in a Ca²⁺-free cell culture medium (J-MEM) containing 1% casein, prior to homogenization in a TEDG buffer (50 mm Tris-maleate buffer, pH 7.4; 1.5 mm EDTA; 2.0 mm DTT; and 10% ($\text{v}\text{v}$) glycerol) containing 0.5 mm phenylmercuric sulfonyl fluoride (PMSF) and 1.0 mm sodium azide. Na₂MoO₄ (final concentration, 20 mm) was added to homogenate fractions which then were incubated at 0–4 °C for 1–5 days with a saturating concentration of ³H-R-1881 (20 nm) in the absence and presence of 2 μm radioinert R-1881. Heparin (200 μg/ml) was added to the incubation medium to “solubilize” nuclear chromatin. Free and bound R-1881 were separated by a hydroxylapatite (HAP) batch procedure. Using these conditions, it has been found that (i) incubation periods of 72–96 h at 0–4 °C were required to achieve maximal specific exchange binding of ³H-R-1881 (B_max) in total homogenates from normal intact rats. Heparin addition (200 μg/ml) did not change B_max and had little or no effect on the rate of exchange. Mean B_max was 6.7 ± 1.6 (SD) pmol ³H-R-1881/mg DNA. R-1881 exchange at 24 h of incubation was only about 40% of B_max. Nonspecific binding, a small fraction (<10%) of B_max, was near maximal at 2 h. Incubation at 15 °C gave similar R-1881 exchange values to those obtained at 0 °C during the first 24 h, but at 48 h and thereafter R-1881 binding markedly decreased. These B_max values in total homogenates of normal intact RVP are about 2.5 times greater than the AR values obtained in 1-day castrated rats, when compared on a DNA basis, (ii) Addition of gelatin (0.25%) to inhibit seminin activity had no effect on B_max. Deletion of either MoO₄^2? or PMSF from the standard exchange medium reduced B_max values ~20%; if both PMSF and MoO₄^2? were deleted, B_max was reduced to a greater extent (~35%). B_max was reduced (40%) when homogenates were prepared without preliminary J-MEM:casein pretreatment and incubated in standard exchange medium with PMSF and MoO₄^2?. (iii) Despite AR stabilization by MoO₄^2? and inhibition of protease activities during exchange incubation, AR inactivation increased exponentially, so that the maximal ³H-R-1881 binding value achieved at 96 h was estimated to represent about 50% of the AR sites originally present. (iv) The binding sites in total homogenates occupied by ³H-R-1881 at 24, 72, and 96 h of exchange exhibited steroid specificity characteristics of AR, as determined by competition studies with a wide variety of steroid hormones and analogues. Scatchard plots of ³H-R-1881 exchange binding in total homogenates of normal intact RVP incubated for 72 or 96 h indicated a single class of affinity sites with apparent K_d of 5 to 6 nm. (v) Sucrose density gradient centrifugation of homogenates incubated for 72 or 96 h showed that the specific ³H-R-1881 binding sites were distributed in two broad peaks associated with low-molecular-weight components. One with S value ~3.5 may be “activated” AR; the other near the top of the gradient (S < 1.6) may include meroreceptor forms of AR.     

Association of gap junctions with endoplasmic reticulum in rat parotid glands

Summary Examination of the parotid gland of the rat has shown specific associations of cisterns of the endoplasmic reticulum with gap junctions. About 20% of the junctions are so intimately associated with cisterns of the endoplasmic reticulum that in freeze fractured material the cisternal membranes remain attached to the junctional membrane faces, obscuring most of the junctional array except for a thin ring of telltale particles. This association was seen only in the parotid gland of the rat, but not that of the other species examined.     

Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum

Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110- A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Ribosome binding to and dissociation from translocation sites of the endoplasmic reticulum membrane

We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC-SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC-SRP complexes can overcome the competition by nontranslating ribosomes.     

Association of poliovirus proteins with the endoplasmic reticulum.

Poliovirus proteins, except P3-7c, are associated with the endoplasmic reticulum after extraction of the cytoplasm and centrifugation of membranes to equilibrium in sucrose gradients. Proteins P3-2, P2-X, and P3-9 are found preferentially among the rough endoplasmic reticulum, whereas P3-7c is located in smooth endoplasmic reticulum fractions. P3-7c is probably not membrane associated, since it can be separated from membranes after centrifugation in buffer. However, P3-4a, P2-5b, P2-X, and P3-9 are avidly bound to membranes and cannot be dislodged with high-ionic-strength buffer containing EDTA or 4 M urea. These proteins are digested by trypsin, indicating peripheral rather than internal localization.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

A rapid, specific protocol for determination of available androgen receptor sites in unfractionated rat ventral prostate cytosol preparations

A modification of the dextran-coated charcoal technique has been successfully employed for the measurement of androgen receptor binding of 5α-dihydrotestosterone in unfractionated rat ventral prostate cytoplasmic extracts. The addition of a small amount of ethanol to the dextran-coated charcoal solution during the adsorption of unbound ligand greatly facilitated charcoal adsorption of ligand associated with low affinity, high capacity binding components and reduced the contribution of the latter cytoplasmic binding components to less than 10 percent of the measured binding at near saturating concentrations, 10 nM, of 5α-dihydrotestosterone. The assay is facile, sensitive, and highly reproducible and a complete saturation curve can be obtained with as little as 100 mg of ventral prostate. This protocol therefore represents a unique procedure for the quantitation and characterization of the cytoplasmic androgen receptor of rat ventral prostate. The concentration of available cytoplasmic androgen receptor in ventral prostate from young mature (80–120 day old) albino rats, 24 hours post orchidectomy, was 10,300 ± 1780 sites per cell and the apparent binding constant for 5α-dihydrotestosterone was 6.49 ± 0.35 × 10⁸ M^?1.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.