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 Kd value of 30 microM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[3H]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 (C8) 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.
Section de Biophysique des Proteines et des Membranes (URA 1290 CNRS), Departement de Biologie Moleculaire et Cellulaire, CEN Saclay, Gif sur Yvette, France.
J J
JJ
Lacapère
J
J
Garin
B
B
Trinnaman
N M
NM
Green
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010777:Photochemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016415:Sequence Alignment; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; C021622:8-azidoadenosine diphosphate