Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes.

Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.     

The interaction of plasma fibronectin with fibroblastic cells in suspension

We have examined the interaction of soluble plasma [3H]fibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4, 22, and 37 degrees C, with half-maximal binding occurring in 12-15 min at 22 degrees C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 +/- 1 X 10(5) sites/cell with an apparent dissociation constant of 8.0 +/- 0.5 X 10(-7) M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [3H]fibronectin is 60-80% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml 44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.     

A specific cyclic guanosine 3':5'-monophosphate-binding protein in Caulobacter crescentus.

A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M.     

Correlation of the 4-5 S form and the 8 S form of the cytosolic androgen receptor in murine skeletal muscle

Binding experiments with the cytosolic androgen receptor from murine skeletal muscle yield with testosterone a biphasic saturation curve and a biphasic Scatchard plot. These binding characteristics result from the conversion of 8 S receptor (KD = 1,4 X 10(-10) M) into 4-5 S receptor (KD = 1,2 X 10(-9) M). This conversion is androgen dependent and is facilitated in vitro by either UV-irradiation or by methods known to activate steroid hormone receptor complexes to a nuclear binding form (e.g. high ionic strength or elevated temperature). The measured data show that both receptor forms are in a complex dissociation equilibrium. The reassociation of the 4-5 S receptor to form the 8 S complex is inhibited by RNase.     

The mechanism of action of vinblastine. Binding of [acetyl-3H]vinblastine to embryonic chick brain tubulin and tubulin from sea urchin sperm tail outer doublet microtubules.

Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.     

Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.     

Binding of human blood-coagulation Factors IXa and X to phospholipid membranes.

A simple centrifugation technique has been developed to study the interaction of human coagulation Factors IXa and X with phospholipid membranes. In the presence of Ca2+, equimolar phosphatidylserine/phosphatidylcholine membranes form tight complexes with Factor X (KD = 2.8 X 10(-8) M); the KD is independent of the phospholipid concentration. Binding sites are available for about 2 mmol of Factor X/mol of phospholipid. Factor IXa has a slightly higher affinity for the phospholipid membrane (KD = 1.2 X 10(-8)M), and competes with Factor X for binding. The experimentally observed competition between Factor X and Factor IXa is in agreement with a model that describes the binding of two distinct ligands to a single class of independent binding sites.     

Radiation-induced crosslinks between carboxylic acids and compounds of biological interest

Various carboxylic acids having short carbon chains (10(-4) or 5 X 10(-4) M) mixed with serum albumin or DNA (1 mg/ml) were irradiated in phosphate buffer solution (10(-3) M, pH 7.0) with 60Co gamma rays. Maleic acid and nicotinic acid showed high binding activity resulting from having an unsaturated double bond and an aromatic ring, respectively. Sodium salts of C18 fatty acids (0.5 mg/ml) such as stearate, oleate, linoleate, and linolenate were also irradiated in the presence of amino acids or nucleobases (10(-4) or 5 X 10(-4) M) in the same buffer solution. The binding yield increased with the increase in the number of double bonds indicating participation of double bonds in the binding.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Receptor-mediated binding of the acute-phase reactant mouse serum amyloid P-component (SAP) to macrophages

Serum amyloid P-component (SAP) is a major acute phase protein of mice which we have previously shown increases the bactericidal activity of elicited, inflammatory macrophages (M phi). The presence of specific receptors for mouse SAP on M phi was demonstrated and the receptor-ligand (SAP) interaction characterized. Purified 125I-labeled mouse SAP binds to elicited M phi with the characteristics of a receptor-mediated event, i.e., the binding was saturable, specific, and reversible. A single type of receptor population was detected with an affinity of 5 x 10(-8) M (KD) and the calculated number of receptor sites per cell was approximately 10(5). Binding of SAP to M phi required Ca2+ or Mg2+ and was inhibited at a pH less than or equal to 5.6. Activated M phi from mice given BCG bind less SAP than nonactivated M phi. Activation of M phi with mouse interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) also decreased their SAP binding capacity. SAP is a glycosylated protein with a high mannose content; therefore mannose and other sugars were tested for inhibition of binding. Specific binding of SAP was inhibited by less than 1 mM concentrations of mannose 6-P, mannose 1-P, and mannose; however, other monosaccharides did not inhibit the binding. Removal of the oligosaccharide from SAP with an endoglycosidase specific for N-linked carbohydrate reduced the binding of SAP to M phi. The pattern of inhibition by sugars, the divalent cation requirement, and the sensitivity to low pH indicate that the receptor binding SAP is the cation-dependent mannose 6-P receptor, or a closely related receptor. The results suggest that SAP may alter or trigger M phi functions associated with inflammation by binding to glycoprotein receptors.     

Alpha-fetoprotein receptors in a human breast cancer cell line

Evidence is presented for the existence of specific receptors for alpha-fetoprotein on the surface of MCF-7 human breast cancer cells. At 4 degrees C, the binding of alpha-fetoprotein to these cells displayed a biphasic saturation curve. Scatchard analysis revealed the presence of at least two binding sites with dissociation constants of 4.5 X 10(-9) M (2,000 sites/cell) and 1.3 X 10(-8) M (135,000 sites/cell), respectively. Binding was inhibited by 85% in the presence of a 5,000-fold excess of unlabeled alpha-fetoprotein and by 50% with the same excess of serum albumin. Competition by other serum proteins was not significant. At 37 degrees C, alpha-fetoprotein was endocytosed and the uptake curve reached a plateau after 3-4 hours of incubation.     

Parathyroid hormone receptors in circulating human mononuclear leukocytes

In this article we demonstrate receptors for parathyroid hormone in circulating mononuclear leukocytes using the radioiodinated analogue (8,18 norleucine, 34 tyrosine) bPTH 1-34 (bovine parathyroid hormone 1-34). Specific binding, which is reversible and saturable, equilibrates within 5 min at 0-4 degrees C with a calculated KD of 8.9 X 10(-11) M. This binding has a pH maximum of 7.0, is magnesium-dependent, and is inversely related to medium calcium concentration. Such binding is completely inhibited by simultaneous addition of 4 ng/ml of bovine parathyroid hormone 1-34, 5 ng/ml of bovine parathyroid hormone 1-84, or 5 ng/ml (8,18 norleucine, 34 Tyr) of 3-34 bPTH, but is unaffected by a biologically inactive parathyroid hormone fragment or other unrelated peptide hormones. Cyclic AMP accumulation increases 3-fold after 5 min exposure of mononuclear leukocytes to bPTH 1-34 in concentrations as low as 1 X 10(-9) M. Lymphocytes appear to be the circulating cells which interact with PTH as indicated by the observations that: 1) lymphocyte-enriched preparations bind three times as much radioligand/cell as do mixed mononuclear leukocytes, 2) monocytes, platelets, granulocytes, and erythrocytes do not bind PTH, and 3) monocytes, but not lymphocytes, degrade the hormone.     

Characterization of norepinephrine binding to plasma proteins of the dog and the rabbit

The binding of 3H-norepinephrine (L-3H-NE, 1.0 X 10(-9) M) to plasma proteins of the dog and the rabbit was studied under controlled conditions. Destruction of NE occurred less rapidly at 22 degrees than at 37 degrees. Binding measured at 22 degrees was equivalent to that at 37 degrees, while binding measured at 0 degree was greater than that at 37 degrees. Therefore, losses of plasma NE were minimized by incubation of samples at 22 degrees for no longer than 30 minutes. L-3H-NE binding was examined in the absence and presence of 10(-9) to 10(-2) M non-labeled L-NE, DL-NE, DL-normetanephrine (NM), DL-epinephrine (E), dopamine (DA), and catechol (C). Specific binding of L-3H-NE varied in the range of NE concentrations (L-3H-NE + non-labeled NE) from 10(-9) M (18.7 +/- 3.1%, rabbit; 25.6 +/- 4.8%, dog) to 10(-6) M (10.8 +/- 3.1%, rabbit; 15.2 +/- 3.6%, dog). Calculated binding constants (KD) were consistent with binding to circulating proteins such as globulins or albumin (4.2 +/- 1.2 X 10(-5) M, rabbit; 5.4 +/- 1.7 X 10(-5) M, dog). In plasma from both species, non-labeled DL-NE, L-NE, E, DA, and C, but not NM (from 10(-9) to 10(-2) M) each significantly displaced L-3H-NE from its binding site in a manner similar to displacement produced by non-labeled NE. The results demonstrate that 1) NE is bound to plasma proteins, although to a lesser extent than had been reported by other investigators; and 2) the binding of catecholamines to plasma proteins may be mediated by the catechol ring.     

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Binding of coagulation factors IX and X to the endothelial cell surface

Bovine coagulation factors IX and X bind to independent sites on bovine aortic endothelial cells. Binding studies with cells maintained serum-free showed that there are at least two classes of binding sites for factor IX and factor X with a dissociation constant of 4.9 x 10(-9) M and 2.1 x 10(-8) M for the respective high affinity sites. Ca+2 was required for specific binding and was reversed by addition of EDTA or EGTA. Competition experiments showed that factor IX and factor IXa bind to the same sites, which are different from the factor X binding sites. Neither binding of factor IX or factor X is inhibited by addition of prothrombin or protein C. Indirect immunofluorescence of factor IX indicated that binding was diffuse on the cell surface.