Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP

Abstract

Abstract2-N[3]SLATP [2-azido-2,3O(1-oxyl-2,2,5,5-tetramethyl 3-carbonylpyrroline) adenosine triphosphate], a photoaffinity spinlabeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotidebinding site stoichiometry of sarcoplasmic reticulum (SR) Ca[2+]ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spinspin interactions between two radicals corresponding to a distance of about 15 å between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca[2+]ATPase is a dimer with the nucleotidebinding sites in close proximity.