title: Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum.
abstract: Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
chemical_list: D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002122:Calcium Chloride; D002118:Calcium