Cross-linking with Concanavalin

Intact cells adsorbed onto concanavalin-A-coated Sephadex or a form of Sephadex cross-linked with glutaraldehyde were used as specific immobilized, cell-surface carbohydrates in the purification of immunoglobulins by immuno-adsorption. °... 

Sephadex (a dextran cross-linked with epichlorohydrin) has been used as an affinity matrix for the separation of [ I]-concanavalin A from the unlabelled lectin. If this technique is successful with other I-labelled proteins, it would help to overcome the uncertainty of whether or not biological properties displayed by labelled materials arise from contamination by the unlabelled material. 

Kokufuta, Zhang and Tanaka developed a gel system that undergoes reversible swelling and collapsing changes in response to saccharides, sodium salt of dextran sulfate (DSS) and a-methyl-D-mannopyranoside (MP) [126]. The gel consists of a covalently cross-linked polymer network of W-isopropylacrylamide into which concanavalin A (ConA) is immobilized. As shown in Fig. 31, at a certain temperature the gel swells five times when DSS ions bind to ConA due to the excess ionic pressure created by DSS. The replacement of the DSS by non-ionic MP brings about collapse of the gel. The transition can be repeated with excellent reproducibility. 

After Zemplen treatment under usual conditions, the sparingly water-soluble hexamers 49 and 51 were obtained in quantitative yields. Their modest solubility in water has however not jeopardized their biological evaluations in solid-phase competitive inhibition assays as well as in their cross-linking abilities with a model plant lectin, Concanavalin A, known to form cross-linked lattices in the presence of multiantennary glycans (39). 

Fig. 2. Schematic representation of Concanavalin A lectin tetramer forming a cross-linked lattice with dimmer 18. (See color plate 1.)...

The studies of mast cell cytokine production described above have shown that maximal induction of cytokine synthesis and release usually occurs in response to IgE-dependent activation. In common with many cell types, there is evidence that FccRI on mast cells is coupled to the phospholipase C effector system that controls two distinct signal transduction pathways, one regulated by Ca " ions and the other by protein kinase C (PKC). Exocytotic degranulation is associated with an increased cytoplasmic level of Ca ions, and activation of mast cells can be therefore achieved by the use of calcium iono-phores which raise intracellular calcium concentrations through a receptor-independent mechanism. Alternative mast cell stimuli include phorbol-12-myristate-13-acetate (PMA) which activates PKC and induces mediator secretion from basophils and rodent mast cells but not from human mast cells, and concanavalin A (Con A), a lectin which can stimulate mast cells by cross-linking of cell-bound IgE and/or cell surface glycoproteins. 

Without doubt, concanavalin A (Con A) is the most celebrated and has proven to be one of the most useful of the plant lectins. Its physical chemical properties and carbohydratebinding properties are well documented in previous reviews [3,8]. Suffice it to note that it was first isolated and crystallized by Sumner and Howell in 1936, who showed it to require metal ions for its activity [74]. By virtue of its interaction with branched a-D-glucans, it is readily prepared by affinity chromatography on crossed-linked dextran (Sephadex) [75, 76]. A homotetramer at pH 7 (subunit M, = 26 500 Da) of Con A has been sequenced [77] and its crystal structure determined both in its native form [38,39] and complexed with methyl a-D-mannopyranoside [40] and Man(al-3)[Man(al-6)]Man[49]. 

An active immobilized form of Penicillium funiculosum dextranase has been prepared by reaction with agarose cyclic imidocarbonate. Conditions have been established for the reaction of dextranase and concanavalin A with glutar-aldehyde to furnish a soluble, cross-linked derivative retaining both enzymic and agglutination properties, although the action pattern of the immobilized form differed from that of the free enzyme. ... 

Phytobaemagglutinins.—Conditions have been established for the modification of concanavalin A with dextranase by a glutaraldehyde-mediated reaction. The soluble, intermolecularly cross-linked conjugate possessed both concanavalin... 

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