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

• Autorzy: Danieluk M. , Bus R. , Pikula S. , Bandorowicz-Pikula J.
• Języki publikacji: EN

Abstrakty

EN

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to bind ATP in vitro with a Kd 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 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 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.

Słowa kluczowe

EN

annexin molecule; triazine dye; Cibacron blue 3GA; annexin VI; ATP-binding domain; dye

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1999
• Tom: 46
• Numer: 2
• Opis fizyczny: p.419-429,fig.

Twórcy

autor

Danieluk M.

• M.Nencki Institute of Experimental Biology, L.Pasteura 3, 02-093 Warsaw, Poland

autor

Bus R.

autor

Pikula S.

autor

Bandorowicz-Pikula J.

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