Rapid filtration study of the phosphorylation-dependent dissociation of calcium from transport sites of purified sarcoplasmic reticulum ATPase and ATP modulation of the catalytic cycle.
Time-resolved filtration measurements using radioactive calcium were conducted to investigate with leaky preparations the kinetic features of the dissociation of transported calcium to the luminal side of the sarcoplasmic reticulum calcium pump, which occurs concomitantly with isomerization of the phosphorylated ATPase. At pH 6 and 20 degrees C, Ca2+ dissociation was moderately fast in the absence of potassium (3-5 s-1 at 0.05 mM ATP), implying that the dephosphorylation step (about 1.5 s-1) was the main contributor to rate limitation under these conditions. Potassium slowed down Ca2+ release but stimulated dephosphorylation, so that in its presence Ca2+-releasing isomerization did contribute to rate limitation, especially at neutral pH. At pH 6 in the absence of potassium and in the presence of magnesium, millimolar concentrations of ATP doubled the rate of Ca2+ dissociation, as also shown by dual-wavelength detection of fast changes in the absorbance of the Ca2+-sensitive dye Antipyrylazo III. Under the same conditions, low-affinity binding of ATP to phosphoenzyme was demonstrated. It is suggested that this low-affinity acceleration by ATP of the crucial step leading to dissociation of transported Ca2+ is the specific interaction responsible for the low-affinity acceleration of overall ATPase activity generally observed in the presence of potassium at neutral pH. Hydrolysis of the Ca2+-deprived phosphoenzyme was accelerated by ATP in the absence but not in the presence of Mg2+ in the dephosphorylation medium. We suggest that metal-free ATP is a more potent activator than Mg X ATP for transitions involving phosphoenzyme.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium