Interaction of carbohydrate binding sites on concanavalin A-agarose with receptors on adipocytes studied by buoyant density method

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Abstract
The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads.