Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N-3-SL-ATP

2-N-3-SL-ATP [2-azido-2 ' ,3 ' -0-(1-oxyl-2,2,5,5-tetram-ethyl-3-carbonyl-p yrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of AIP with a nitroxide moiety attached to the ribose ring and an azido gro up attached to C2 of the adenine ring, was used to study the nucleotide-bin ding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-AT-Pase. The la bel 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 spin-spin interactions betw een two radicals corresponding to a distance of about 15 Angstrom between l abels bound to adjacent sites on the enzyme. This indicates that the minima l functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.