Interactions of saccharides with concanavalin A. Relation between calcium ions and the binding of saccharides to concanavalin A.

Previous studies (KALB, A. J., AND LEVITZKI, A. (1968) Biochem. J. 109, 669; SHOHAM, M., KALB, A. J., AND PECHT. I. (1973) Biochemistry 12, 1914) were interpreted as showing that transition metal and calcium ions must be bound to concanavalin A before this lectin can bind sugars. In contrast, we find that the addition of calcium ions fails to affect the following properties of manganese concanavalin A if the metalloprotein is prepared in their presence: (a) ESR spectrum of the manganese ion in the protein; (b) spin-lattice relaxation time of solvent water protons measured in the presence of the protein: and (c) spin-lattice and transverse relaxation times of the i*C carbons of cr-methyl-D-glucopyranoside (uniformly enriched with 14 % l*C) in the presence of manganese concanavalin A. These results indicate that saccharide binding activity of the protein is independent of calcium ion binding if a transition metal ion (Mn2+) initially binds to the protein in the presence of calcium ions. The role of calcium ion binding to concanavalin A appears to be that of accelerating the rate of formation of the final transition metal-protein complex as observed by Barber and Carver (BARBER, B. H., AND CARVER, J. P. (1973) J. Biol. Chem. 248, 3353). charides and polysaccharides possessing nonreducing terminal a-o-mannopyranoside or /?-o-glucopyranoside residues (1). The biological properties of this protein have been reviewed recently (2). The physical properties of Con A’ have been studied extensively, including determination of its structure by x-ray diffraction techniques (3, 4). Con A exists as a dimer between pH 3.5 and 5.6 with a molecular weight of 54,000; tetramers form at higher pH (5, 6). Each monomeric unit of the protein possesses a site for transition metal ions (Si) and a site for calcium ions (82). Kalb and coworkers (7, 8) suggested that both sites must be occupied before saccharide binding activity can occur. Recently, Barber and Carver (9) measured the spin-lattice relaxation value (Ti) of solvent water protons in the presence of Mnz+free Con A and observed the effect of adding manganese ions and calcium ions to the solution. Their results suggest that calcium ions exert a cooperative effect in binding manganese ions to the Si site by accelerating the rate of refolding of the protein about the transition metal ion. In this paper, we report on the relationship between calcium ions and the binding of saccharides to Con A. In contrast to previous reports (7, 8), our results indicate that saccharide binding activity of Con A is independent of calcium ion binding to the protein, provided that the Si site is fully occupied by a transition metal ion.