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 led to purification of three protein bands at 68, 36 and 35 kDa from bovine heart that required Ca2+ for binding. 2. Polyacrylamide-immobilized phosphatidylserine separated this fraction into a phospholipid-binding part (68 kDa, 35 kDa), also attaching to phospholipid vesicles even in the presence of calcyclin, and a flow-through part, constituting approx 30% of the total fraction (36 kDa). 3. Enzyme assays and electrophoretic 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 sialoglycoproteins and depended strongly on the integrity of carboxyl and hydrophobic groups of the enzyme. The interaction between the two proteins had a KD value of 110 nM. 4. Application of annexin-specific antibodies revealed an immunological relationship of the 35 and 68 kDa calcyclin-binding proteins to members of the annexin family, namely to annexin II (35 kDa) and annexin VI (68 kDa). The N-terminal amino acid sequence of a cleavage peptide of the 68 kDa protein was identical to a sequence stretch in human annexin VI, corroborating this evidence.