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Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca2+-ATPase by Competitive Inhibition of [γ-32P]TNP-8N3-ATP Photolabeling

Springer Nature,

Volume 1377, 2016

DOI:10.1007/978-1-4939-3179-8_22, Dimensions: pub.1002560561, PMID: 26695037,

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  1. (1) Aarhus University, grid.7048.b, AU
  2. (2) University of Cape Town, grid.7836.a

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Denmark

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Description

The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

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2016: Blocked

Research area: Medicine

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2016: Level 1

Research area: Medicine

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