BIOCHEMICAL-CHARACTERIZATION OF A CA2-DEPENDENT LECTIN FROM THE HEMOLYMPH OF A PHOTOSYMBIOTIC MARINE BIVALVE, TRIDACNA-DERASA (RODING)()

A Ca2+-dependent lectin was purified from the hemolymph of a photosymb iotic bivalve, Tridacna derasa, An electrophoretically homogeneous for m was obtained by using affinity chromatography with Sepharose 4B. Mor e than 80% of the hemolymph protein was accounted for by this lectin. The apparent molecular mass of the lectin, in its native form exhibiti ng hemagglutinating activity, was estimated by gel filtration analysis to be approximately 480 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in the absence of reductants, it migrated as a s ingle band corresponding to a very large size, while in the presence o f 2-mercaptoethanol, it migrated as two distinct bands of 23 and 46 kD a. These results indicate that the subunits were linked by disulfide b ridges to form the native protein, After reducing pyridylethylation, e ach of the 23- and 46-kDa polypeptides was isolated by gel filtration in a mobile phase containing guanidine-HCl. The two polypeptides had t he same amino-terminal sequence and a similar amino-acid composition, and in the presence of 2-mercaptoethanol gave a single band on isoelec tric focusing at a pH of 6.0. The results suggested that the 46-kDa pe ptide is a homodimer of 23-kDa subunits held together by a covalent bo nd other than a disulfide linkage, This lectin required calcium ions f or its activity. By ultraviolet spectrophotometry the association cons tant for the calcium ion was determined to be 0.88 mM, The hemagglutin ating activity decreased dramatically below pH 6.5, but re-increased t o the original level when the solution was neutralized, Such a pH-depe ndent alteration of the ligand-binding activity was similar to that fo und in vertebrate asialoglycoprotein receptors. The amino-terminal seq uence, determined to 29 residues, showed homology to some Ca2+-depende nt, C-type, animal lectins. This observation, together with the calciu m ion requirement, suggests that the tridacnid lectin is a member of t he C-type lectin family.