IDENTIFICATION OF AMINO-ACID-RESIDUES PHOTOLABELED WITH 8-AZIDOADENOSINE 5'-DIPHOSPHATE IN THE CATALYTIC SITE OF SARCOPLASMIC-RETICULUM CA-ATPASE

The photoreactive ADP analogue 8-N3-ADP binds in the dark to the catalytic site of the sarcoplasmic reticulum Ca-ATPase. An apparent K(d) value of 30 muM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[H-3]ADP in the presence of calcium results in irreversible inhibition of ATPase activity with corresponding stoichiometries of covalently and specifically photolabeled Ca-ATPase. The site of photolabeling of the Ca-ATPase in the presence of calcium has been explored. Controlled trypsin digestion of the labeled protein shows that 8-azido-ADP is incorporated in the B subfragment. Extensive trypsin digestion of the labeled protein releases a small peptide as revealed by gel filtration chromatography (Sephadex G-50). Further HPLC purification on a reverse-phase column (C-8) eluted with a water/acetonitrile gradient buffered at pH 6 or at pH 2 gives a single labeled peptide. Edman degradation of that isolated peptide, as well as the amino acid composition, shows that it contains five amino acid residues (Val-530-Arg-534) with the radioactivity localized on Thr-532 and Thr-533.