Inhibition and labeling of the Ca2(+)-ATPase from sarcoplasmic reticulum by periodate oxidized ATP.
The analog of ATP obtained by oxidation of the ribose ring of ATP with periodate (oxATP) was used as a reagent for the inhibition and labeling of the Ca2(+)-ATPase purified from sarcoplasmic reticulum membranes. The substrate concentration dependence for hydrolysis showed a biphasic pattern for both ATP and oxATP as substrates. Preincubation of Ca2(+)-ATPase in the presence of 0.05 mM CaCl2, 5 mM MgCl2, 100 mM KCl and oxATP led to an irreversible inhibition. This inhibition occurred faster at alkaline pH. The presence of ADP, adenyl-5'-imidodiphosphate (AMP-PNP) or EGTA in the preincubation medium decreased the rate of inhibition. OxATP covalently labels the enzyme: the labeling was decreased by ADP. This ADP-protected labeling increased with time until it reached approx. 1 mol [3H]oxATP per mol ATPase. The rate of labeling of the ADP-protected group correlated with the rate of loss of ADP-protected activity. Trypsin digestion of oxATP-labeled ATPase followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that fragment A1 contained a high degree of label that is displaced by ADP. We propose that the A1 fragment is situated close to the ribose ring when the adenosine moiety of ATP is bound to the catalytic site of the Ca2(+)-ATPase.
Mignaco
J
J
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Cidade Universitaira, Brasil.
Scofano
H M
HM
Barrabin
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000345:Affinity Labels; D010446:Peptide Fragments; C004282:periodate-oxidized adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases