3′-Arylazido-8-azido ATP—A cross-linking photoaffinity label for ATP binding proteins

Abstract Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high- affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM- CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.

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Isotope-Coded ATP Probe for Quantitative Affinity Profiling of ATP-Binding Proteins

Analytical Chemistry ◽

10.1021/ac401415z ◽

2013 ◽

Vol 85 (15) ◽

pp. 7478-7486 ◽

Cited By ~ 19

Author(s):

Yongsheng Xiao ◽

Lei Guo ◽

Yinsheng Wang

Keyword(s):

Binding Proteins ◽

Atp Binding

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Molecular Weight Determination of DNA-Binding Proteins by UV Cross-linking Combined with SDS Polyacrylamide Gel Electrophoresis

A Laboratory Guide to In Vitro Studies of Protein-DNA Interactions ◽

10.1007/978-3-0348-7561-5_7 ◽

1991 ◽

pp. 79-91 ◽

Cited By ~ 1

Author(s):

Melya J. Hughes ◽

Haimin Liang ◽

Jean-Pierre Jost

Keyword(s):

Molecular Weight ◽

Dna Binding ◽

Gel Electrophoresis ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Dna Binding Proteins ◽

Polyacrylamide Gel ◽

Cross Linking ◽

Molecular Weight Determination

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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.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4174 ◽

1999 ◽

Vol 46 (2) ◽

pp. 419-429 ◽

Cited By ~ 1

Author(s):

M Danieluk ◽

R Buś ◽

S Pikuła ◽

J Bandorowicz-Pikuła

Keyword(s):

Binding Site ◽

Binding Proteins ◽

Direct Sequencing ◽

Nucleotide Binding ◽

Atp Binding ◽

Dependent Manner ◽

Cibacron Blue ◽

Annexin Vi ◽

Cibacron Blue 3Ga ◽

Triazine Dye

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.

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