Peroxidase binding to cell-bound Concanavalin A

The relationship between the amount of Concanavalin A bound to a cell surface and the amount of peroxidase which binds to the lectin was investigated. It was found that only a few lectin molecules are revealed by the enzyme and that this number is dependent on the cell type.     

High affinity divalent cation binding to actin. Effect of low affinity salt binding

Monomeric actin labeled with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS-actin) displays a fast fluorescence intensity increase immediately upon addition of salt and then a slow fluorescence intensity change concurrent with Ca2+/Mg2+ exchange at the high affinity divalent cation binding site on actin. The fast change appears to reflect competitive binding of K+ at low affinity (nonspecific) sites and of Mg2+ or Ca2+ at low and intermediate affinity sites. Binding of cation at the low affinity sites (but apparently not at the intermediate affinity sites) results in an increase in k-Ca and k-Mg and thus a decrease in affinity for divalent cations at the high affinity site. The effect of Mg2+ on k-Ca is twice that of K+ for equal fractional saturations of the low affinity binding, and the effect of K+ and Mg2+ together on k-Ca reflects competitive binding at the low affinity sites. Thus the affinity and kinetics of divalent cation binding at the high affinity site of actin are significantly affected by concurrent cation binding at low affinity sites.     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

High affinity binding of cholecystokinin to small cell lung cancer cells

The binding of 125I-Bolton Hunter-cholecystokinin octapeptide (125I-BH-CCK-8) to small cell lung cancer cell lines was investigated. 125I-BH-CCK-8 bound with high affinity (Kd = 2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific 125I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease.     

High affinity binding of paclitaxel to human serum albumin.

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle-related toxicity, we developed a novel, water-soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel-albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP-HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 x 10(6) M(-1)). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 x 10(6) M(-1) and K2 = 1.0 x 10(5) M(-1)). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.     

Calorimetric study of carbohydrate binding to Concanavalin A

Flow microcalorimetry has been used to examine the ΔH of binding of two types of saccharides, a series of simple monosaccharides and a series of α-(1 → 4)-linked glucosides, to the lectin Concanavalin A. It has been found that the ΔH decreases with any change in the stereochemistry of a hydroxyl group relative to methyl α-d-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The ΔH's of binding for the α-(1 → 4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for α-(1 → 4)-linked glucopyranosides, in contrast to that proposed for α-(1 → 2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.     

High affinity thyroxine binding to purified rat liver plasma membranes.

Two sets of high-affinity thyroxine binding sites (K_D 0.39 ± 0.06 nM and 23 ± 5 nM) were detected on purified rat liver plasma membranes. Thyroxine is bound with high stereospecificity regarding iodine substituents and alanine side chain modifications of the molecule. Thyroxine binding is inhibited by -SH blocking agents and proteases. The highest affinity thyroxine binding site is also affected by phospholipase A and is distinct from triiodothyronine binding sites present in the membrane preparations; arguments are given for its plasmalemma origin.     

High affinity GnRH binding to testicular membrane homogenates

The binding of GnRH to membrane homogenates from collagenase-dissociated normal rat interstitial cells is shown to be of high affinity and specific. The dissociation constant for the radioligand used, [¹²⁵I-Tyr⁵, D-Ala⁶, N^αMeLeu⁷, Pro⁹-NEt]-GnRH is 0.26 ± 0.02 nM and the number of binding sites is 17 fmoles/mg membrane homogenate. There is one GnRH antagonist whose affinity is greater for the pituitary than for the testis. This antagonist has the same affinity for testis homogenates as for ovarian homogenates.     

A fluorimetric study of the lanthanides binding to Concanavalin A

• 1.1. The bincling of Tb³⁺ and other lanthanides to Con A has been studied by sensitized Tb³⁺ luminescence, by quenching of intrinsic fluorescence and by activity measurements.
• 2.2. In all the experimental conditions tested, it was found that holo and apo Con A bind lanthanide ions at a site different from the bincling sites of the constitutive metals, Mn²⁺ and Ca²⁺.
• 3.3. The bound lanthanide did not affect the saccharide bincling ability and the hemoagglutinating ability of Con A.
• 4.4. The intrinsic fluorescence of Con A is quenched by the bincling of Tb³⁺ and Gd³⁺. The same quenching is obtained by shifting the pH of Con A from pH 6.5 to 4.5.
• 5.5.It is proposed that H⁺ and Ln³⁺ completely quench a tryptophan, perhaps the residue 88 or 182.

     

High affinity binding of human and bovine thrombins to p-chlorobenzylamido-epsilon-aminocaproyl agarose.

Human thrombin (EC 3.4.21.5) binds tightly to p-chlorobenzylamido-epsilon-aminocaproyl agarose, and is not eluted by 2 M NaCl at pH 8. Its zymogen, human prothrombin, does not bind to the same absorbent. 2 M NaCl partially elutes DFP-treated thrombin. For native human and bovine thrombins, protein and activity are quantitatively eluted by 25% dioxane, and upon rechromatography the active human enzyme exhibits the same binding properties. Equally tight binding of human thrombin occurs with derivatives of the m- and p-chlorobenzylamines. With the o-chloro derivative or benzylamine itself insolubilized to epsilon-aminocaproyl agarose, thrombin is eluted by high ionic strength. Bovine trypsin and bovine factor Xa bind less tightly than thrombin to p-chlorobenzylamido-epsilon-aminocaproyl agarose, being eluted by high ionic strength. It is proposed that the specific thrombin adsorption is related to a secondary binding site of high affinity and with hydrophobic properties. This site is not available in the zymogen. Furthermore, the less specific protease, trypsin, and the more specific protease, factor Xa, lack this binding site.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Concanavalin A inhibition of alpha-bungarotoxin binding to a nonfusing muscle cell line.

Incubation of a nonfusing muscle cell line, BC3H1, with concanavalin A (Con A) results in a maximum decrease of 35% in the cell's ability to bind alpha-bungarotoxin (alpha-BuTx). The Con A-induced inhibition of 125I-alpha-BuTx binding is reversible and the degree of inhibition parallels the degree of saturation of Con A binding sites on the cell surface. The maximum level of Con A-induced inhibition of 125I-alpha-BuTx binding is not affected by increasing the time of incubation in Con A, using higher concentrations of Con A or by increasing the time of incubation in the presence of 125I-alpha-BuTx. In addition, all BC3H1 cells in culture are sensitive to the Con A-induced inhibition of 125I-alpha-BuTx binding. A comparison of the pseudo-first order rate constants for 125I-alpha-BuTx binding to untreated (8.6 x 10(4) M-1 S-1) and Con A-treated (5.4 x 10(4) M-1 S-1) BC3H1 cells, however, shows that those acetylcholine receptors in Con A-treated cells which bind 125I-alpha-BuTx do so with a lowered apparent affinity. Partial inhibition of toxin-binding capacity is not a consequence of two classes of acetylcholine receptors on the cell surface. Furthermore, individual receptors experience partial inhibition of their binding capacity by Con A, resulting in receptors with at least one binding site blocked and at least one site available for alpha-BuTx binding.     

Concanavalin A binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca2+ content of the cells. 45Ca2+ fluxes were measured under conditions where artifacts due to Ca2+ binding to concanavalin A could be excluded. Both 45Ca2+ influx into and efflux from these cells are significantly activated by the lectin. If 45Ca2+ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in 45Ca2+ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 micrograms/ml) no significant increase in 45Ca2+ influx occurs but an increase in 45Ca2+ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca2+ influx into the cells and that activation of Ca2+ efflux occurs as a response to an increase in the cytosolic Ca2+ activity. Thus, Ca2+ may well play a role in triggering lymphocyte activation.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

Concanavalin A binding to brain particulate preparations and its effect on adenylate cyclase.

[³H] -Concanavalin A binding to brain particulate preparations measured by a filtration technique was found to show a characteristic regional specificity with the caudate-putamen area exhibiting the greatest density of concanavalin A (con A) binding sites. The synaptic membranes were shown to be the most highly enriched of the subcellular fractions examined in terms of lectin-binding glycoproteins. Con A was also shown to inhibit the basal adenylate cyclase activity of cerebral, cerebellar, and caudate-putamen particulate preparations in a concentration-dependent manner. This lectin sensitivity of the adenylate cyclase is apparently an intrinsic property of the enzyme complex since a detergent dispersed preparation of the cerebral cortical enzyme was equally inhibited by con A. It is proposed that one of the membrane con A binding sites in brain tissue is a component of the adenylate cyclase system.