title: Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]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.
authors.affiliation: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, 8000, Aarhus C, Denmark. jdc@fi.au.dk.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.affiliation: Institute of Infectious Diseases and Molecular Medicine, Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, South Africa.
authors.forename: David B
authors.initials: DB
authors.lastname: McIntosh
authors.affiliation: Institute of Infectious Diseases and Molecular Medicine, Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, South Africa.
authors.forename: David G
authors.initials: DG
authors.lastname: Woolley
authors.affiliation: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, 8000, Aarhus C, Denmark.
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D010761:Phosphorus Radioisotopes; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D013194:Staining and Labeling; D014301:Trinitrobenzenes / Q000737:chemistry
chemical_list: D010761:Phosphorus Radioisotopes; D014301:Trinitrobenzenes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
keywords: ATP binding affinity; Ca2+ -ATPase mutants; P-type ATPase family; Photoaffinity labeling; Sarcoplasmic reticulum Ca2+ -ATPase; [γ-32P]TNP-8N3-ATP synthesis