Interaction between prolactin and rabbit mammary prolactin receptor in the presence of environment-modifying agents

The binding assay of prolactin (PRL) to the receptor in the rabbit mammary gland was carried out with varying concentrations of NaCl, KCl, CaCl2, MgCl2, glycerol, glucose, sucrose and urea. The agents did not affect the binding capacity. The ionic bond-breaking agents (NaCl and KCl) had little effect on changes in the association rate constant (k+1) of PRL binding to the receptor and the dissociation rate constant (k-1) of bound PRL. The inclusion of other agents changed the k+1 and the k-1. Among the agents examined, chaotropic salts (CaCl2 and MgCl2) inhibited the binding of PRL greatly, and were the most effective in decreasing the k+1. Both hydrogen- and hydrophobic bonds are involved in the interaction between PRL and the receptor. The data suggest that hydrophobic bonding is primarily an important force participating in the binding of PRL to its receptor.     

Opiate receptor binding of naloxone in the rat brain: effect of sodium ions

The binding levels and opiate receptor binding parameters were determined for 3H-naloxone in rat brain in the presence of NaCl added in vitro. An addition of NaCl at concentrations of 5-35 mM to the reaction medium caused an increase in the level of the antagonist receptor binding. The maximal level of 3H-naloxone reception activation was observed in the presence of 10-20 mM NaCl and was, on the average, 25%. Both the increase in the NaCl dose in vitro and its decrease caused a gradual diminution of the Na+ effect. An analysis of opiate receptor saturation with 3H-naloxone revealed that the label interacted with one type of the binding sites irrespective of NaCl concentration. The affinity of receptor binding sites for 3H-naloxone increased already at NaCl concentration of 2.5 mM. In contrast, the apparent maximal number of binding sites did not change after NaCl addition at concentrations which coincided with the intracellular Na+ level but was decreased with an increase (up to 50-100 mM) in NaCl present in the reaction mixture. The results obtained point to the existence of two different binding sites that are coupled with the 3H-naloxone reactive opiate receptor.     

Rat ovarian and adrenal prolactin receptors. Sizes and effects of divalent metal ions

Receptor fractions were prepared from follicle-rich ovaries (for FSH), luteal cell-rich ovaries (for LH and PRL), and adrenals (for PRL) of rats. Divalent metal ions, Mg++, Ca++, and Mn++ showed inhibitory effects on the binding of LH and FSH to their receptors. The binding of the former was more sensitive to these ions than the latter. On the other hand they showed bell-shaped promotive effects on PRL-ovarian receptor binding, the maximal effects being observed at 10-20 mM. Besides these ions, Ba++ also had a promotive effect, while other divalent metal ions such as Zn++, Cd++, Ni++, and Co++ showed inhibitory effects on PRL-ovarian receptor binding at 5 mM. Mg++ and Ca++ also promoted PRL-adrenal receptor binding, while Mn++ promoted the binding at 10 mM but inhibited it at higher concentrations. Association constant (Ka) and binding capacity (Bmax) of PRL receptors of the ovary and the adrenal were significantly different (ovary: Ka = 0.69 X 10(10) M-1, Bmax = 62 fmol/mg protein, adrenal: Ka = 0.21 X 10(10) M-1, Bmax = 99 fmol/mg protein). Ka of the ovarian PRL receptor was not influenced by these divalent ions, while that of the adrenal receptor was doubled by Ca and Mn ions, Bmax of the latter was also increased. A cooperative effect of Mg and Ca ions was observed on Ka and Bmax of the adrenal receptor. The sizes of the PRL binding sites of these organs revealed by affinity labelling were 17K and 40K in the ovary, and 40K and 110K in the adrenal. These results indicate the different properties of receptors in these different target organs.     

Monoclonal antibody detection of prolactin-binding subunits in the rabbit mammary gland.

The structure of prolactin (PRL) receptor in the rabbit mammary gland was examined using a receptor-specific monoclonal antibody (MAb). The PRL receptor preparation used was purified by making use of a PRL-affinity column. MAb inhibited the binding of PRL to the receptor, in a dose-dependent manner and completely at a high concentration. Using the receptor directly labelled by 125I, the preparation was incubated with MAbs and the immune complex was collected by Pansorbin and examined by SDS/polyacrylamide-gel electrophoresis. The autoradiography showed that three species with apparent Mr values of 77,000, 41,000 and 25,000 specifically reacted with MAbs. The pattern changed little in the presence or absence of dithiothreitol. Western blot analysis showed that two species (Mr 77,000 and 41,000) reacted with MAb. Affinity labelling of the receptor with labelled PRL revealed three bands with Mr values of 96,000, 60,000 and 43,000 on SDS gels. The high-Mr complex (Mr greater than 200,000) was always present at the top of the gel. These results show that the mammary gland contains at least three PRL-binding subunits. The differences in Mr before and after PRL binding were close to the Mr of PRL. This would suggest that each PRL binding subunit reacts with one PRL molecule.     

Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein.

Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range (>/= 5 mM MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure. The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.     

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Mutational analysis of the ligand-binding domain of the prolactin receptor

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partial Proteolytic Digestion of the Mammary Prolactin Receptor: Identification of Smaller Prolactin Binding Fragments

Abstract

Partial proteolytic digestion of the mammary prolactin (PRL) receptor was used to generate receptor fragments and analyze their immunoreactivity and PRL binding properties. Tryptic digestion of the PRL receptor produced two immunoreactive fragments (Mr ≈ 30,000 and ≈ 15,000) that reacted with a monoclonal anti-PRL receptor antibody and still specifically bound PRL, while the complete immunoreactive PRL binding unit (Mr ≈ 42,000) disappeared. Neither chymotrypsin nor V8 protease were able to generate any immunoreactive receptor fragments. These receptor fragments may represent smaller PRL binding receptor form(s) of biological significance.     

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

Binding and structural characteristics of a soluble lactogen-binding protein from rabbit mammary-gland cytosol.

Specific receptors for prolactin (PRL) are known to be present on plasma membranes and intracellular membranes of mammary gland. We now report, however, the detection and characterization of a soluble lactogen-specific binding protein in high-speed (200,000 g) cytosolic preparations from pregnant- and non-pregnant-rabbit mammary gland. The binding protein was not detectable by poly(ethylene glycol) precipitation; instead, bound and free 125I-labelled human growth hormone (hGH; a potent lactogen) was separated using a mini-gel filtration technique. Specific binding of 125I-hGH reached an apparent equilibrium between 10 and 14 h at 21-23 degrees C. It was dependent on mammary-gland protein concentration and, partially, on Ca2+ or Mg2+ concentrations. Scatchard analysis revealed steep curvilinear plots, the high-affinity component having a KA of approximately 3 X 10(10) M-1. Gel filtration on calibrated Ultrogel AcA34 columns of 125I-hGH-cytosol complexes or of cytosol alone, followed by measurement of 125I-hGH binding in each eluted fraction, indicated that the binding protein had an Mr of 33,000-43,000. A specific binding protein of the same size was observed when 125I-ovine or -human PRL, but not 125I-bovine GH, was used as ligand. The apparent lactogenic specificity was confirmed by a lack of cross-reactivity of the binding protein with an anti-[GH receptor (rabbit liver)] monoclonal antibody. Polyacrylamide-gel electrophoresis of 125I-hGH covalently cross-linked to cytosol with disuccinimidyl suberate revealed binding proteins of Mr 35,000 (non-reduced) and 37,000 (reduced), results comparable with those obtained by gel filtration and indicating an absence of inter-subunit disulphide bonds. These studies have shown the presence of an apparently naturally soluble lactogen-binding protein in the cytosolic fraction of rabbit mammary gland. The relationship between this binding protein and the membrane PRL receptor is not yet known.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr ? 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.     

The melanoma growth stimulatory activity receptor consists of two proteins. Ligand binding results in enhanced tyrosine phosphorylation.

The human melanoma growth-stimulatory activities (MGSA alpha, beta, gamma/GRO) are products of immediate early genes coding for cytokines that exhibit sequence similarity to platelet factor-4 and beta-thromboglobulin. MGSA/GRO alpha has been demonstrated to partially complete for binding to the approximately 58-kDa neutrophil receptor for another beta-thromboglobulin-related chemotactic protein, IL-8. We demonstrate that when [125I]MGSA/GRO alpha was cross-linked to receptors/binding proteins from human placenta, there were two major [125I]MGSA cross-linked bands of approximately 64,000 and approximately 84,000 Mr. Because [125I]MGSA exists primarily in monomer and dimer forms at the concentrations used here, it is not clear whether the receptor/binding proteins represented by the cross-linked bands are approximately 50,000 and approximately 70,000 or approximately 58,000 and approximately 78,000 Mr. Ligand binding to the receptor proteins is associated with enhanced tyrosine phosphorylation of a number of substrates, including proteins in the same Mr range as the MGSA/GRO receptor/binding proteins.     

The liver angiotensin receptor involved in the activation of glycogen phosphorylase

Specific angiotensin binding to rat hepatocytes and purified liver plasma membranes was measured by using biologically active [(3)H]angiotensin (sp. radioactivity 14Ci/mmol). The kinetic parameters for angiotensin binding to hepatocytes are: K(+1) (association rate constant). 100mum(-1).min(-1); K(-1) (dissociation rate constant), 2min(-1); K(d) (dissociation constant). 30nm; maximal binding capacity, 0.42pmol/10(6) cells or 260000 sites/cell. Angiotensin binding to membranes is profoundly affected by GTP (0.1mm) and NaCl (100mm); these regulatory compounds greatly enhance both the rate of association and of dissociation and also the extent of dissociation. K(d) amounts to 10nm in the presence of GTP+NaCl and to 1.5nm in their absence; maximal binding capacity is 0.70pmol/mg of protein, both with or without GTP+NaCl. The relative affinities of 11 angiotensin structural analogues were deduced from competition experiments for [(3)H]angiotensin binding to hepatocytes and to membranes (in the latter case, GTP + NaCl were not included, in order to study the higher affinity state of the receptor). These are highly correlated with their biological activity (activation of glycogen phosphorylase in hepatocytes). Binding to membranes occurs in the same concentration range as the biological effect. On the other hand, the existence of numerous spare receptors is suggested by the observation that binding of the agonists to hepatocytes requires 25-fold higher concentrations than those needed for their biological activity. These data clearly suggest that the detected binding sites correspond to the physiological receptors involved in the glycogenolytic action of angiotensin on rat liver.     

Effects of sodium and temperature on naloxone binding in brain tissues of a urodele amphibian

1. Partially purified brain membranes obtained from male rough-skinned newts (Taricha granulosa) were used to determine the effects of NaCl and temperature on the specific binding of the opioid receptor antagonist [3H]naloxone. 2. The addition of NaCl to the incubation medium at concentrations up to 400 mM produced a dose-related increase of the specific binding of [3H]naloxone. 3. The addition of other salts to the incubation medium had less pronounced effects: KCl and MgCl2 slightly increased and decreased, respectively, the specific binding of naloxone, and CaCl2 had no effect. 4. Results of an equilibrium saturation experiment showed that the addition of 200 mM NaCl resulted in over a 10-fold increase in the number of high affinity (KD = 0.61 nM) binding sites for naloxone, with no changes in the number of low affinity (KD = 21.8 nM) binding sites. 5. Changes in NaCl concentrations did not significantly affect either dissociation constant. 6. The binding of [3H]naloxone was temperature-dependent; it increased when the incubation temperatures were elevated from 0 degree C to 37 degrees C. 7. Results obtained for this urodele amphibian are compared with those available for other vertebrate species.     

Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane

Solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the human erythrocyte membrane (Wolf, H.U., Dieckvoss, G. and Lichtner, R. (1977) Acta Biol. Ger. 36, 847) has been phosphorylated and dephosphorylated under various conditions with respect to Ca2+ and Mg2+ concentrations. In the range, 0.001--100 mM, the rate of phosphorylation was dependent on Ca2+ concentration, showing a maximum at 10 mM. The phosphorylation rate was nearly independent of the Mg2+ concentration within the range 0.01-1 mM. This enzyme has at least three Ca2+ binding sites with different affinities and regulatory functions: (1) binding to the high-affinity site yields phosphorylation of the enzyme; (2) binding to a low-affinity site (Ca2+ concentrations higher than 40 microM) inhibits dephosphorylation or the conformational change which is necessary for dephosphorylation; (3) by binding to an additional low-affinity site, Ca2+ at concentrations higher than 1 mM abolishes negative cooperative behaviour (shown below 1 mM Ca2+) and causes weak positive cooperativity between at least two catalytic subunits in the phosphorylation reaction. The phosphoprotein obtained at Ca2+ concentrations above 1 mM dephosphorylates spontaneously after removal of the divalent metal ions. Addition of Mg2+ accelerates the dephosphorylation rate. Affinities of the inhibitory Ca2+ binding sites are reduced by the binding of substrate or K+.     

Differences in the characteristics and distribution of rat luteal receptors for native and deglycosylated human choriogonadotropin.

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.