Carbohydrate-binding activity of concanavalin A containing various numbers of calcium and manganese ions

After treatment with EDTA, fragment-free concanavalin A (Con A), F3, was fractionated into three major fractions, f1, f2, and f3, by CM-cellulose chromatography. Fully metallized fragment-free Con A, F3M, was prepared by incubation of F3 with excess metal ions and also fractionated into f1M, f2M, and f3M by the same method as for F3. The Con A preparations obtained were analyzed for metal content by atomic absorption spectrophotometry and for number of carbohydrate-binding sites by ultraviolet absorbance change on binding of p-nitrophenyl alpha-D-mannopyranoside (PNP . Man) to Con A. Both f1 (Ca: 0.2, Mn: 0.1) and f1M (Ca: 0.4, Mn: 0.2) had less than 0.6 carbohydrate-binding sites per tetramer of Con A. f2 (Ca: 2.1, Mn: 0.9) and f2M (Ca: 2.1, Mn: 1.9) had 1.9 and 1.8 carbohydrate-binding sites, respectively. f3 (Ca: 4.1, Mn: 2.1) and f3M (Ca: 3.8, Mn: 4.0) had 4.1 and 4.2 carbohydrate-binding sites, respectively. Carbohydrate-binding sites of Con A were in a stoichiometric relation to bound Ca2+. The molar absorptivity change at 317 nm was 2 X 10(3) M-1 . cm-1. Precipitation activity of the above Con A preparations towards glycogen was also estimated using glycogen labeled covalently with Remazolbrilliant Blue R. The precipitation activity of Con A as well as PNP . Man-binding was strongly affected by the number of bound Ca2+. A slight contribution by Mn2+ was also detected in the precipitation activity.