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

Abstract

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.

A review of TNP-ATP in protein binding studies: benefits and pitfalls

“…This pump hydrolyzes ATP (but not TNP-ATP) and binds ATP, ADP, and AMP with differing affinities. TNP analogs of all three nucleotides (i.e., TNP-ATP, TNP-ADP, and TNP-AMP) have been used to bind SERCAs and lock them into different conformations associated with their hydrolysis and pumping cycles (for reviews, see ( 119 , 120 ); see also ( 121 , 122 , 123 , 124 )).…”