IDENTIFICATION OF ANNEXIN-II, ANNEXIN-VI AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AS CALCYCLIN-BINDING PROTEINS IN BOVINE HEART

1. Matrix-immobilized calcyclin as affinity ligand in chromatography l ed to purification of three protein bands at 68, 36 and 35 kDa from bo vine heart that required Ca2+ for binding. 2. Polyacrylamide-immobiliz ed phosphatidylserine separated this fraction into a phospholipid-bind ing part (68 kDa, 35 kDa), also attaching to phospholipid vesicles eve n in the presence of calcyclin, and a flow-through part, constituting approx 30% of the total fraction (36 kDa). 3. Enzyme assays and electr ophoretic mobility showed an at least close relationship of the 36 kDa band to glyceraldehyde-3-phosphate dehydrogenase. Interaction between enzyme and calcyclin in a solid-phase assay was inhibited by sialogly coproteins and depended strongly on the integrity of carboxyl and hydr ophobic groups of the enzyme. The interaction between the two proteins had a K(D) value of 110 nM. 4. Application of annexin-specific antibo dies revealed an immunological relationship of the 35 and 68 kDa calcy clin-binding proteins to members of the annexin family, namely to anne xin II (35 kDa) and annexin VI (68 kDa). The N-terminal amino acid seq uence of a cleavage peptide of the 68 kDa protein was identical to a s equence stretch in human annexin VI, corroborating this evidence.