Vicia graminea lectin binding to human M and N erythrocytes

The mode of binding of Vicia graminea¹²⁵I-labelled lectin to human M and N erythrocytes at 4°C has been investigated. The labelled lectin retained the full activity of native lectin. Lectin association at 4°C was characterized by a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 3 to 5 min, reaching steady-state within 15 min. Incubation of cells for 15 min at 4°C with increasing concentrations of Vicia graminea¹²⁵I-labelled lectin showed that saturation binding occurred. Scatchard analysis of equilibrium data determined over a wide range of lectin concentrations yielded a curvilinear plot with an upward concave slope; this representation indicated that there was not a single homogeneous class of noninteracting binding sites. This result could indicate two or more independent classes of binding sites or one class of interacting sites exhibiting negative cooperativity. Since unlabelled lectin, which at the concentration used, rapidly binds to available receptors, did not affect the dissociation rate of the labelled lectin and since identical Scatchard plots were found using native and formaldehyde-fixed erythrocytes we conclude that there are two classes of independent Vicia graminea binding sites on human erythrocytes. Computer analysis of the Scatchard plots gave high- and low-affinity constant (7.07±1.1) · 10⁷ M^?1 and (0.2±0.01) · 10⁷ M^?1, respectively, for N erythrocytes and (1.13±0.18) · 10⁷ M^?1 and (0.24±0.01) · 10⁷ M^?1, respectively for the M cells. N erythrocytes were estimated to have 0.085 · 10⁵ high-affinity and 2.1 · 10⁵ low-affinity sites and M erythrocytes, 0.011 · 10⁵ high affinity and 0.13 · 10⁵ low-affinity sites. N cells therefore have 10-times as many sites as M cells. Studies of the dissociation of ¹²⁵I-labelled lectin from N and M cells in the presence of unlabelled lectin gave dissociation rate constants of 51 · 10^?4 s^?1 and 1.97 · 10^?4 s^?1 for the high- and low-affinity sites of N cells and 13 · 10^?4 s^?1 and 1.6 · 10^?4 s^?1 for the high- and low-affinitym sites of M cells, indicating that the binding of Vicia graminea lectin to human erythrocytes is reversible.     

Vicia villosa B4 lectin is the second anti-Tn lectin shown to react better with blood group N than M antigen

Earlier studies showed thatMoluccella laevis lectin, which has anti-Tn specificity, reacts more strongly with native or desialylated blood group N glycophorin A than with the respective glycophorins of blood group M. We now present results indicating thatVicia villosa B4 anti-Tn lectin, which does not show detectable reaction with untreated glycophorins or erythrocytes, reacts better with desialylated blood group N antigen than with asialo M antigen. This was demonstrated by three assays: (1) agglutination of asialoerythrocytes; (2) binding of biotinylated lectin to asialoerythrocytes immobilized on ELISA plates; and (3) inhibition of lectin binding to asialo-agalactoglycophorin with asialoglycophorins M and N. These results supply further support for the conclusion that glycophorin of blood group N has more GalNAc residues unsubstituted with Gal (Tn receptors) than glycophorin of blood group M.Abbreviations GPA glycophorin A - GPA-M and GPA-N GPA from OM and ON erythrocytes, respectively - MLL Moluccella laevis lectin - PBS 0.02m phosphate buffer/0.15m NaCl, pH 7.4 - PNA peanut agglutinin - RBC erythrocytes - TBS 0.05m Tris buffer/0.15m NaCl, pH 7.4 - TBS-T TBS containing 0.02% Tween 20 - VVL Vicia villosa B4 lectin     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Properties of enucleated cells. III. Changes in cytoplasmic architecture of enucleated BHK21 cells following trypsinization and replating.

At low concentrations of concanavalin A (conA), binding of the lectin to the erythrocytes appears to be the rate-limiting step in the agglutination of these cells. At higher concentrations of lectin the rate of agglutination is concentration-independent, indicating that the aggregation reaction is rate-determining. Only 5 to 7% of the 1.2 × 10⁵ receptor sites need be occupied by con A in order for agglutination to take place. Although trypsin-treated cells bind 30% less ¹²⁵I-conA, they agglutinate better than untreated cells. At high lectin concentrations, erythrocyte agglutination by wheat germ agglutinin (WGA) is more than 8 times faster than the conA-mediated reaction. Lowering of the temperature to 0 °C reduces the rate but not the extent of the agglutination by both lectins. Mechanical shear reduced the conA-mediated agglutination of native cells by more than 160-fold and that of trypsinized and neuraminidase-treated cells 6-fold and 4-fold, respectively.It is concluded that metabolic activity, receptor mobility (i.e. cluster or patch formation) and cytochalasin B-sensitive processes, all of which have been reported to be involved in the lectin-mediated agglutination of fibroblasts and other cells, do not play a role in erythrocyte agglutination. Lectin-mediated erythrocyte agglutination appears to be governed primarily by the rate and extent of binding of lectin to the cell surface, the cell surface charge (modifiable by enzyme treatments or polycations) and the shear forces in the suspension. Morphological studies confirm and amplify these conclusions.     

Characterization of the Galactose-Specific Binding Activity of a Purified Soluble Entamoeba histolytica Adherence Lectin

ABSTRACT. We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffnity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4°C by radioimmunoassay; the dissociation coefficient (Kd was 2.39 × 10^?8 M^?1 with 5.97 × 10⁴ lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 × 10⁵/cell) rather than a significantly reduced dissociation constant (4.93 × 10^?8 M^?1). At 4°C, there was no measurable Gal-specific binding of the adherence protein to the Lec and IdlD.Lecl CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 μg/ml) to CHO cells was equivalent at 4°C and 37°C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 μg/ml). Gal-specific binding of the lectin at 4°C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells. In summary, these findings indicate that the purified E. histolytica adherence lectin demonstrates saturable Gal-specific binding to 1–6 branched-N-linked and not O-linked galactose terminal cell surface carbohydrates; however, apparently only a small percentage of purified amebic lectin molecules actually possess galactose binding activity.     

The lack of mitogenic response of neuraminidase-treated and untreated human blood lymphocytes to divalent, hexavalent, or insoluble Helix pomatia a hemagglutinin

The mitogenic effects on human blood lymphocytes of Helix pomatia A hemagglutinin (HP) were measured by assaying incorporation of [¹⁴C]thymidine into cellular DNA. This highly purified lectin binds to human lymphocytes, treated with neuraminidase, but not to untreated lymphocytes. HP, which is hexavalent in its native form, totally failed to induce DNA synthesis in neuraminidase-treated as well as in untreated cells. Divalent HP, prepared by partial reduction and alkylation, and HP insolubilized by coupling to nylon sheets, also lacked mitogenicity. Control experiments indicated that neuraminidase-treated lymphocytes responded well to mitogenic lectins such as PHA. The lack of mitogenicity of HP was in contrast to the effects of soy bean agglutinin (SBA), which resembled HP in regard to carbohydrate specificity and ability to bind to neuraminidase-treated lymphocytes only. SBA was strongly mitogenic for neuraminidase-treated lymphocytes. The mitogenic effects of SBA were not inhibited by including varying doses of HP in the incubation mixtures. The results indicate that binding of lectin to carbohydrate receptors on the lymphocyte surface is not by itself sufficient to trigger DNA-synthesis.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.     

Interaction of synthetic glycopeptides carrying clusters ofO-glycosidic disaccharide chains (β-d-Gal(1–3)-α-d-GalNAc) withβ-d-galactose-binding lectins

The specificity of severald-galactose-binding lectins including Agaricus bisporus (mushroom),Arachis hypogaea (peanut),Bauhinia purpurea andVicia graminea has been examined by inhibition of hemagglutination using a series of synthetic oligopeptides representing the N-terminal end of glycophorin A from N and M individuals, all carrying one or several disaccharide chains,d-Galβ1–3-d-GalNAcα-(T-hapten). Peanut lectin was inhibited by T-hapten-carrying glycopeptides, but the presence of a cluster of disaccharide chains had no effect on the lectin specificity. On the contrary, bothAgaricus bisporus andBauhinia purpurea lectins exhibited an enhanced reactivity with polyglycosylated peptides suggesting that their combining site might include two proximal galactose residues. All synthetic glycopeptides inhibitingVicia graminea lectin carry a cluster of T-disaccharide chains and the leucine residue at the N-terminal end, and the presence of a Glu residue at position 5 slightly increased the lectin activity. It is concluded that the binding ofVicia graminea is dependent upon a specific spatial conformation including a cluster of T-hapten chains in close vicinity of a hydrophobic surface represented by an appropriate N-terminal amino acid residue.     

Fractionation of human lymphocytes with plant lectins. I. Structural and functional characteristics of lymphocyte subclasses isolated by an affinity technique using Lens culinaris lectin.

We have fractionated human peripheral blood lymphocytes (PBL) into subclasses with an affinity technique that takes advantage of the specific interaction between the plant lectin, Lens culinaris agglutinin (Lentil-PHA), and its cell surface receptors. PBL were incubated in plastic tubes or petri dishes coated with a gelatin layer to which lentil-PHA had been coupled using a water soluble carbodiimide compound. Adherent cells were recovered by melting the gelatin, and bound lectin was removed by exposing the cells to an appropriate sugar hapten. Approximately 25 to 50% of PBL specifically adhered to gelatin-coated tubes derivatized with lentil-PHA. Binding studies with ¹²⁵I-labeled lentil-PHA showed that, compared to unfractionated PBL which bound 2.0 × 10⁶ lentil-PHA molecules per cell, adherent cells bound more (3.7 × 10⁶/cell), and nonadherent cells bound less (1.1 × 10⁶/cell) lentil-PHA. By contrast, there were no differences among these groups for binding of other plant lectins. When stimulated in vitro by optimal mitogenic concentrations of lentil-PHA, lymphocytes adherent to lentil-PHA responded better than their nonadherent counterparts whereas the two groups responded the same to stimulation by the leukoagglutinin from Ph. vulgaris. The data indicate that plant lectins may be used to isolate PBL subclasses with different structural and functional properties.     

Human Erythrocyte Receptor of l-Fucose-specific Lectin Produced by Streptomyces sp.

L-Fucose-specific lectin produced by Streptomyces no. 16-3 (SFL 16-3) was labeled with N- succinimidyl-[2, 3-³H]-propionate to quantitatively investigate its binding to human erythrocytes. The binding inhibition by sugars was competitive, and 5mM L-fucose or 20 mM d-mannose completely inhibited the binding. Among plant lectins, Lotus tetragonolobus, Ulex europeus I, soybean and wheat germ lectin showed competitive inhibition. The association constant and the average number of binding sites for human blood group O erythrocytes were approximately 3 × 10⁷ M^-1 and 1 × 10⁶ cell^-1, respectively. Trypsinization of erythrocytes preferentially increased the number of binding sites for human A and B erythrocytes but not for O erythrocytes.

Membrane components were extracted from human B and O erythrocytes and their binding activity for SFL 16-3 was tested using the hemagglutination-inhibition assay. Poly(glycosyl)-ceramide was the predominant receptor and its fucosyl residue was essential for binding. The crude glycoprotein fraction showed only slight inhibition activity.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

Rapid degradation of [3H]leucine-enkephalin by intact neuroblastoma cells.

Enkephalin, a brain peptide with morphine-like activity, is rapidly degraded by N4TG1 neuroblastoma cells which contain opiate receptors. The enzymic activity is temperature dependent and it is maximal at 37°C with an apparent Km of 5 × 10^?5M. The enzyme can be inhibited by bacitracin and puromycin with apparent Ki values of 3.2 × 10^?5M and 2.3 × 10^?7M, respectively. Digesting intact cells with trypsin greatly diminishes the ability of the cells to degrade enkephalin and suggests that the degradative enzyme is probably localized at the cell surface. Opiates such as morphine and naloxone as well as leu- and met-enkephalin and D-Ala²-substituted analogs at very high concentrations (10^?5M) have no effect upon enzymic activity despite the fact that they totally block binding of the labeled enkephalin to receptors. The data strongly suggest that there is no correlation between receptor occupancy and rate of enkephalin degradation.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B₄ lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8×10⁵ M^?1 at 4°C, whereas that for GalNAcα-1-phosphate was 1.3×10⁵ M^?1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B₄ lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

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 α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/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 (K_d) 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 (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/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 α-methyl-D-mannopyranoside.     

Interaction of concanavalin A with differentiated and undifferentiated murine neuroblastoma cells.

Tritium-labeled acetyl-concanavalin A (³H-Con A) was used to study its kinetics of binding at 0 °C to murine neuroblastoma cells (clone neuro 2-A) grown in the differentiated (monolayer) and Undifferentiated (spinner) states. The binding of ³H-Con A to both cell types gives sigmoidal saturation curves, suggesting positively cooperative binding of the lectin. The Hill coefficient is 1.75 for differentiated and 1.36 for Undifferentiated cells. The maximal number of ³H-Con A molecules bound per cell is 2.3 × 10⁷ and 3.4 × 10⁷ for differentiated and Undifferentiated cells, respectively, and the apparent rate constants for formation of the lectin-cell complex are 6.13 × 10², m^?1, s^?1 for the Undifferentiated and 6.68 × 10², m^?1, s^?1 for the differentiated cells. The lectin bound to spinner cells does not dissociate spontaneously to any measurable extent over a 60-min period at 0 or 37 °C, but the lectin-cell complex dissociates rapidly after addition of α-methyl-d-mannopyranoside. At 37 °C, this sugar causes virtually complete dissociation of the cell-lectin complex within 30 min. The ³H-Con A dissociated from spinner cells is indistinguishable from the original ³H-Con A by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, gel filtration through Bio-Gels P-30 and P-100, and specific binding to spinner cells. Both the original and the dissociated ³H-Con A are dimers at pH 7.4. The sugar-induced dissociation of the labeled lectin from spinner cells is not accompanied by shedding or inactivation of the lectin binding sites of the cell surface.