Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca 2+- and membrane-binding proteins, has been shown to bind ATP in vitro with a K-d in the low micromolar concentration range. However, this protein doe s not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by c hanges in AnxVI affinity to Ca2+ in the presence of ATP. Using limited prot eolytic digestion, purification of protein fragments by affinity chromatogr aphy on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI w as located in a C-terminal half of the AnxVI molecule. To further study Anx VI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their acti vities. We have observed that AnxVI binds to CB3GA immobilized on agarose i n a Ca2+-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in s olution, evoking reversible aggregation of protein molecules, which resembl es self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-bin ding protein.