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At least 19 recordsLinked to original sources
PubMed1983

The concanavalin A receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A.

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using 125I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13 . 10(6) M-1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83 . 10(6) M-1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.

Agglutination↗
PubMed1979

Effect of concanavalin A dose, unbound concanavalin A, temperature, Ca2+ and Mg2+, and vinblastine on capping of concanavalin A receptors of human peripheral blood lymphocytes.

The capping of Concanavalin A (Con A) receptors induced by Con A was studied using human peripheral blood lymphocytes. The effects of Con A dose (5--100 micrograms/ml), pretreatment at 4 degrees C, unbound Con A, extracellular Ca2+ and Mg2+ and vinblastine were evaluated using Con A-horseradish peroxidase and electron microscopy. Lymphocytes incubated with Con A at 4 degrees C and fixed with glutaraldehyde exhibited Con A-horseradish peroxidase around the entire cell periphery. After raising the temperature to 37 degrees C, the Con A-horseradish peroxidase moved to form a cap at one pole of the cell and subsequently underwent endocytosis. Capping of Con A receptors induced by Con A at 37 degrees C was observed only at low Con A concentrations in the presence of unbound Con A and extracellular Ca2+ and Mg2+. Increased capping was found after pretreatment of cells with Con A at 4 degrees C, removing unbound Con A and/or removing extracellular Ca2+ and Mg2+, and by treatment with vinblastine. Following removal of both unbound Con A and extracellular Ca2+ and Mg2+, the percentage of capped cells at 37 degrees C was the same as on pretreatment at 4 degrees C under the same conditions. While pretreatment at 4 degrees C caused the breakdown of microtubules, removal of unbound Con A and/or extracellular Ca2+ and Mg2+ had no morphological effect on microtubules or microfilaments. Following exposure of lymphocytes to vinblastine and removal of unbound Con A, capping of Con A receptors by Con A was observed in over 90% of cells at all Con A dosages. However, when cells were exposed to vinblastine in the presence of unbound Con A the formation of Con A caps was either partially or completely inhibited.

Calcium↗
PubMed1976

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.

Animals↗
PubMed1983

Different effects of concanavalin A on insulin binding to cultured cells in monolayers: "blocking" and "trapping" effects of concanavalin A.

The effect of concanavalin A on insulin binding was studied in cells cultured in a monolayer. Preliminary incubation of concanavalin A with Reuber rat hepatoma cells ( R117 - 21B ) showed an inhibitory effect on insulin binding at 25 degrees C and 4 degrees C; a "blocking" effect. But, even at high concentrations (greater than 50 micrograms/ml), concanavalin A could not completely inhibit the specific binding of insulin to these cells. Under these conditions, the number of high affinity insulin-binding sites decreased when compared to the number of sites in the control. In contrast, when cells first were incubated with insulin then with insulin plus concanavalin A, the binding of insulin increased, even at high concentrations of the lectin. This is a "trapping" effect. This effect increased the number of insulin-binding sites as compared to the number in the control. The apparent equilibrium constant of insulin for the cells was changed slightly by the concanavalin A in R117 - 21B cells. The binding of concanavalin A to these cells also was investigated. Insulin did not affect its binding to the cells. This study shows that insulin-binding sites can be classified into two groups in which insulin binding is affected by concanavalin A or is independent of the lectin. The "blocking" effect of concanavalin A on insulin binding suggests that the binding sites of insulin and concanavalin A on the insulin receptors are very close.

Animals↗
PubMed1983

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation.

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.

Blood Platelets↗
PubMed1976

Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments.

With normal rat kidney cells in monolayer culture, we have studied the distribution on the cell surface of receptors for concanavalin A, and the distribution of the smooth muscle myosin-like protein inside the same cell, using specific fluorescence microscopic methods. The concanavalin A receptors were initially uniformly dispersed over the cell surface, but 20 min after the addition of concanavalin A at 37 degrees, the receptors showed a variety of nonuniform surface distributions, including extended parallel linear arrays. These arrays of receptors were found to be superimposed on the linear arrays of the intracellular myosin-containing filaments, indicating that a transmembrane linkage of the receptors and the filaments had occurred. This linkage required a lateral redistribution of concanavalin A receptors, since it did not occur with succinylated concanavalin A, but was subsequently induced if the cells that had been reacted with succinylated concanavalin A were then treated with antibodies to concanavalin A. The redistributions of concanavalin A receptors on the surfaces of these normal rat kidney cells, however, were much less extensive than the patching that was induced on the surfaces of the same cells infected with, and transformed by, Rous sarcoma virus.

Cell Line↗
PubMed1985

Effects of bovine serum albumin on the interaction of concanavalin A and succinyl-concanavalin A with phospholipid bilayers.

Under physiological conditions, concanavalin A interacts with the surface of phospholipid liposomes through two distinct classes of binding sites, a relatively small number of high affinity sites and a much larger number of lower affinity sites. Addition of bovine serum albumin induces extensive additional binding of concanavalin A to liposomal membranes and this binding is saturable and "specific" (alpha-methyl mannoside inhibitable). Fraction V and high purity albumin both induce almost identical levels of concanavalin A binding to liposomes. Scatchard plots of the binding data demonstrated the induction of a large number of new, relatively high affinity lectin-binding sites on addition of albumin. Albumin-induced binding of concanavalin A to the bilayer surface shows a broad pH optimum and is not inhibited by 40% (w/v) ethylene glycol, suggesting that hydrophobic forces are relatively unimportant. In contrast, divalent succinyl-concanavalin A shows very little tendency to bind to liposomes, either in the absence or presence of albumin. Passage of high purity albumin down a concanavalin A affinity column or treatment with periodate completely eliminates the additional lectin binding. It thus seems likely that albumin-induced concanavalin A binding to liposomes is related to the presence of a concanavalin-A-binding component. This phenomenon may have important implications for lectin-binding studies carried out on membranes which have been exposed to serum proteins.

Binding Sites↗
PubMed1976

Carbohydrate analysis of concanavalin A-reactive and concanavalin A-nonreactive mycobacterial polysaccharides.

Concanavalin A-nonreactive polysaccharide and 2 concanavalin A-reactive polysaccharides of differing concanavalin A affinities that had been purified from culture filtrates of Mycobacterium tuberculosis were subjected to carbohydrate analysis by gas and gas-liquid chromatography. Concanavalin A-nonreactive polysaccharide was found to be D-arabinogalactan, and the concanavalin A-reactive polysaccharide of highest affinity was found to be D-arabinomannan. The lower affinity concanavalin A-reactive polysaccharide was rich in arabinose and probably represented a mixture, with more than one polysaccharide present. Glucan was found to be a significant contaminant of all 3 polysaccharides.

Arabinose↗
PubMed1989

A jack bean protein similar to pea seed ferritin and unrelated to concanavalin A is the only iron storage protein in jack bean seeds, although concanavalin A can form ferritin-like iron cores in vitro.

Iron storage in living organisms is performed by ferritins. These proteins are built up from 24 subunits organized in a spherical shell forming a coat to a core of bioavailable iron. Recently, it was observed that concanavalin A from jack bean can form in vitro iron cores similar to those of animal ferritins (Yariv, J., Kalb, A. J., Helliwell, J. R., Papiz, M. Z., Bauminger, E. R., and Nowik, I. (1988) J. Biol. Chem. 263, 13508-13510). From this observation and from the comparison of the three-dimensional structures of horse spleen ferritin and of the form of concanavalin A which forms soluble polynuclear iron complexes, these authors suggested that concanavalin A is the apoferritin of jack bean seeds. On the basis of immunological and biochemical results, we report here that a protein purified from jack bean seeds, unrelated to concanavalin A and similar to plant seed ferritins, is responsible for iron storage in jack bean seeds. Furthermore, concanavalin A does not contain iron in vivo. Therefore we conclude that a unique protein, ferritin, stores iron in vivo in jack bean seeds and that concanavalin A provides an unusual model for studying the formation of iron cores inside a protein shell.

Amino Acids↗
PubMed1980

Bovine plasma amine oxidase interactions with concanavalin A in solution and with concanavalin A-Sepharose.

The reaction of bovine plasma amine oxidase, a glycoprotein, with Concanavalin A in 0.1 M potassium phosphate buffer, pH 7.0 at 25 degrees C were investigated by equilibrium and kinetic methods. A tentative mechanism for the reaction was derived. The Concanavalin A-enzyme interaction was used to show that the carbohydrate is not essential for activity and that the carbohydrate is covalently attached to the protein at a site distant from the active site. Concanavalin A-Sepharose 4B affinity chromatography of the enzyme was found to be useful for obtaining the pure enzyme. Chromatographic conditions which elute the enzyme are reported. The superiority of the Concanavalin A-Sepharose 4B over that of the substrate-affinity Sepharose affinity support is discussed. The phenylhydrazone derivative of the enzyme, which was inactive, was shown to bind to the concanavalin A-Sepharose column.

Animals↗
PubMed1976

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.

Binding Sites↗
PubMed1983

Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes.

Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A-Sepharose have been carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 x 75 cm columns of concanavalin A-Sepharose and a 4 x 400 ml gradient (Varigard) with 0.5 M methyl alpha-D-glucopyranoside as limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins have been determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility, and charge density, is a useful parameter in the chromatographic separation of protein from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein was tightly bound by concanavalin A-Sepharose. Subsequent experiments showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of concanavalin A-reactive residues for strong binding. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the molecular weights of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with concanavalin A-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl alpha-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).

Ammonium Sulfate↗
PubMed1983

Release of cell-associated concanavalin A by methyl alpha-D-mannopyranoside reveals three binding states of concanavalin-A receptors on mouse fibroblasts.

Based on the partial reversibility of concanavalin A binding by saturating concentrations of methyl alpha-D-mannopyranoside (MeManp) three states of cellular association could be characterized: type I, most rapidly established and most tightly bound, not released by MeManp at 0 degrees C or at 37 degrees C; type II, most loosely bound, released by MeManp at 0 degrees C, therefore not critically dependent on the temperature at which the release reaction is performed; type III, intermediate strength of binding, released by MeManp only at 37 degrees C, thus reflecting the temperature-sensitive nature of these cell complexes. A similar temperature dependence was found for cell-bound concanavalin A when it was displaced by an excess of the same lectin. The types of binding are seen irrespective of the temperature at which the cellular association was established. About 10% of the concanavalin A molecules bind to receptor structures in a saturable way and represent the type I association. Type I association is clearly distinct from types II and III. Type II seems to be the precursor of type III. Most of the type II associations can be converted into type III associations since ConA-cell complexes originally sensitive to the action of MeManp at low temperature gradually do become resistant after prolonged association at the same temperature. The temperature dependence in binding to cells was, however, not related to receptor mobility since glutaraldehyde-treated cells had most of the release properties that were observed in untreated cells and which did not markedly differ between 3T3 cells and their SV40-transformed counterparts. In contrast to cellular binding, dissociation of concanavalin A from Sephadex beads by saturating concentrations of MeManp was complete irrespective of temperature.

Animals↗
PubMed1975

A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells. Relationship between concanavalin A binding and cell agglutination.

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregatesmas predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates. The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [3-H]concanavalin A revealed that the binding to approx;0% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.

Agglutination Tests↗
PubMed1987

Glycoprotein-concanavalin A interaction: role of protein conformation in the specific interaction of glycoprotein with concanavalin A.

The specific binding of two glycoproteins, ovomucoid and ovalbumin to concanavalin A was studied in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl and 1 mM each of magnesium acetate, calcium acetate and manganese chloride at 30 degrees C. Native ovalbumin was a better ligand than ovomucoid in its reaction with concanavalin A-sepharose 4B; in the inhibition of the lectin binding to dextran and cross-linked dextran, Sephadex G-150, and in the rate of precipitin reaction. Reduction of disulphide bonds of the two glycoproteins with 2-mercaptoethanol and subsequent alkylation with iodoacetate produced significant conformational change as evidenced by the increase in the hydrodynamic volume measured by intrinsic viscosity. The native and modified glycoproteins were indistinguishable in inhibiting the binding of dextran and Sephadex G-150 to the lectin, however, their pronase digest (or glycopeptides) were most effective inhibitors. Substantial (4-fold) decrease in the rate of precipitin reaction of the lectin with glycoprotein which was noted with conformationally altered glycoproteins suggested the importance of native protein conformation in the specific interaction of the two glycoproteins with concanavalin A.

Alkylation↗
PubMed1991

Interactions of concanavalin A with glycoproteins. A quantitative precipitation study of concanavalin A with the soybean agglutinin.

Certain oligomannose-type glycopeptides have been previously shown to be bivalent for binding to concanavalin A and capable of precipitating the lectin by forming homogeneous cross-linked lattices [L. Bhattacharyya, M. I. Khan, and C.F. Brewer, Biochemistry, 27 (1988) 8762-8767]. In the present study, the effect of protein environment on the binding properties of an oligomannose-type oligosaccharide has been examined through quantitative precipitation analysis of the interactions of concanavalin A (Con A) with the soybean (Glycine max) agglutinin (SBA), which is a tetrameric glycoprotein possessing a single Man9-oligomannose chain per monomer. The results showed that SBA forms two different types of cross-linked complexes with tetrameric Con A, depending on the relative ratio of the two molecules in solution. At a concentration of one equivalent or less, SBA forms a 1:1 complex with Con A. At concentrations exceeding one equivalent, SBA forms a 2:1 complex with Con A. However, SBA forms only 1:1 cross-linked complexes with dimeric forms of Con A, such as acetyl- and succinyl-Con A. The results demonstrated that the total valency of the carbohydrate of SBA is a function of both the quaternary structure of Con A, as well as the relative ratio of SBA to Con A. In addition, the individual Man9-oligosaccharide, which as a glycopeptide is bivalent for binding to Con A, expresses univalency when present on the protein matrix of SBA.

Carbohydrate Sequence↗
PubMed1982

Insulin-like activity of photochemically obtained monovalent monomeric concanavalin A in the presence of anti-concanavalin A antibodies: dependence on multivalency for stimulation of glucose oxidation of rat fat cells.

Insulin-like action of monovalent monomeric concanavalin A (m-Con A) was examined in rat adipocytes in the presence of anti-m-Con A antiserum. The antisera from rabbits injected with m-Con A reacted with not only monovalent monomeric but also tetravalent tetrameric concanavalin A (alpha-Con A) in Ouchterlony double diffusion analysis. m-Con A alone did not show any appreciable effect on glucose oxidation of adipocytes while it slightly inhibited glycerol release stimulated by epinephrine. In contrast, exposure of adipocytes to m-Con A in the presence of antibodies to m-Con A resulted in stimulation of glucose oxidation and inhibition of epinephrine-stimulated lipolysis. The stimulation and the inhibition with m-Con A in the presence of the antibodies were of the same degree as those with alpha-Con A. Both alpha- and m-Con A were slightly active in inhibiting 125I-labeled insulin binding. These results demonstrate that the ability of anti-m-Con A antiserum to aggregate m-Con A bound to receptors on the isolated-adipocyte plasma membrane allowed m-Con A to mimic the biological activity of insulin and that the aggregation of receptors for ligands other than insulin can induce insulin-like action in rat fat cells.

Adipose Tissue↗