Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase.
We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover.
Davidson
G A
GA
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, Republic of South Africa.
Berman
M C
MC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics
D004533:Egtazic Acid; D007811:Lanthanum; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium