CARBOHYDRATE-BINDING PROPERTIES OF THE HEMOLYTIC LECTIN CEL-III FROM THE HOLOTHUROIDEA CUCUMARIA-ECHINATA AS ANALYZED USING CARBOHYDRATE-COATED MICROPLATE

The carbohydrate-binding properties of the hemolytic lectin CEL-III fr om the Holothuroidea Cucumaria echinata were studied using the micropl ate assay system which we have recently developed [Hatakeyama et al. ( 1996) Anal. Biochem. 237, 188-192], When the binding of CEL-III to lac tose covalently immobilized on a microplate was examined using colloid al gold solution, the binding was detected with as little as 1 mu g/ml protein, Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for C EL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temper atures revealed that the affinity is higher in the acidic pH region an d at lower temperatures. From the Ca2+-dependence profile for the bind ing of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3 mM, These results suggested that the carbohydrate-binding properties of CEL-III are closely relate d to its hemolytic activity, although an additional interaction betwee n the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.