T. Palm et al., Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N-3-SL-ATP, BIOL CHEM, 382(3), 2001, pp. 417-423
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.