Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Modulation of thymocyte membrane potential by concanavalin A.

Binding of convanavalin A, a potent mitogenic lectin, to thymocyte surface membrane causes depolarization of membrane potential. The effect is suppressed by alpha-methyl-D-glucoside, a haptenic inhibitor of this lectin, or by low temperature. Colchicine and cytochalasin B aslso suppress the change. These data indicate that perturbation of thymocyte surface membrane receptors induced by concanavalin A might be linked to change in the functional state of cellular cytoskeletal systems in turn causing depolarization of thymocyte surface membrane. The initial event generated by receptor-ligand interaction on the outer surface could be translated into cellular interior action only under highly fluid conditions of membrane lipid. Depolarization of thymocyte plasma membrane may be involved in the triggering mechanism of metabolic burst associated with blastoid transformation.     

Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (～6 × 10⁶/cell equivalent) low-affinity sites (K_a = 1.3 × 10⁵ M^?1) and (b) fewer (～0.4 × 10⁶/cell equipment) specific receptors (K_a = 6.8 × 10⁶ M^?1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of～1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concanavalin A-binding induces multimerization of the 55,000 D glycoprotein.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Role of the membrane concanavalin A binding site in platelet-fibrin interactions

Concanavalin A was employed to study the role of platelet membrane glycoproteins in platelet-fibrin interactions during clot formation. A rheological technique was used to study the interactions, measuring the clot rigidity and platelet contractile force simultaneously during the formation of network structure. Concanavalin A lowered the clot rigidity and contractile force of a platelet-rich plasma clot by a small extent. Plasma glycoproteins probably compete with platelet membranes for concanavalin A binding in platelet-rich plasma. Both native concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) lowered the clot rigidity and contractile force of a washed platelet-fibrin clot dramatically, almost down to those values found for fibrin clots. Inhibition studies with alpha-methyl-D-mannoside indicated that the concanavalin A effects were specific for the concanavalin A binding capacity to platelets. The effects of native concanavalin A on platelet-fibrin clots were only partially reversible, while the succinyl concanavalin A effects were completely reversible. The observed concanavalin A effects are probably mainly due to concanavalin A binding to platelet membrane glycoproteins. The concanavalin A binding site appears to play an important role in the fibrin binding to platelets.     

Cooperative binding of concanavalin A to thymocyte membrane vesicles augments uptake of alpha aminoisobutyrate.

(1) Membrane vesicles from rabbit thymocytes accumulate alpha-aminoisobutyrate in the presence of 0.1 M NaCl. Uptake is 1/2 maximal after about 2 min and reaches a plateau value (61 pmoles/mg protein) after 30 min. (2) Up to 25 mug concanavalin A/ml, binding of the lectin describes a sigmoid curve indicative of a cooperative process. (3) At lectin concentrations up to 8 mug/ml, lectin binding enhances the uptake of alpha-aminoisobutyrate (maximally 30%).     

Spontaneous local membrane curvature induced by transmembrane proteins

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally.Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature ($\approx 0.036$ nm^?1) whereas AQP0 causes a comparably small negative curvature ($\approx ?0.019$ nm^?1).Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.     

Periodate induced cross-linking of concanavalin A-reactive membrane proteins of rabbit thymocytes.

Purified plasma membranes of rabbit thymocytes are exposed to sodium periodate and galactose oxidase at conditions similar to those used to induce mitogenic transformation of lymphocytes. The membrane proteins are then fractionated by dodecyl sulfate poly-acrylamide gel electrophoresis. At concentrations of 0.005 M, Na IO4 cross-links 55,000 D and 110,000 D glycoproteins which are known to specifically bind concanavalin A. Galactose oxidase has a similar cross-linking effect, but, at the same time causes proteolytic degradation of membrane proteins. Our data indicate that oxidizing agents, like NaIO4 and galactose oxidase, can indeed cross-link receptors of the thymocyte plasma membrane as has often been proposed as a possible mechanism of their action.     

Selectivity in heavy metal- binding to peptides and proteins

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.     

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells.

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the I-220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Metal ion binding to concanavalin A

Metal ion activation of saccharide binding has been studied for concana-valin A near pH 7.0. Although two metal ions, a transition metal ion and a Ca²⁺ ion, can bind, both are not required. Ca²⁺ alone, Mn²⁺ alone, or Ca²⁺ with other transition metal ions can activate this lectin. Only one Ca²⁺ ion per subunit or only one Mn²⁺ per subunit is sufficient. Metal ion binding was studied by magnetic resonance techniques and direct binding assays. Saccharide binding activity was monitored by following the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside. When Ca²⁺ binds to demetalized concanavalin A, the transition metal ion site is hindered. When Mn²⁺ alone binds to demetalized concanavalin A, saccharide binding activity is induced. A subsequent conformational change, not necessary for carbohydrate binding activity, covers the Mn²⁺.