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 does 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 changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-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 activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-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 solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.
Danieluk
M
M
Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, Warszawa, Poland.
Buś
R
R
Pikuła
S
S
Bandorowicz-Pikuła
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D017317:Annexin A6 / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D013552:Swine; D014227:Triazines / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D017317:Annexin A6; D014227:Triazines; C012842:Cibacron Blue F 3GA; D000255:Adenosine Triphosphate; D002118:Calcium