The experiments described indicate that heat is released when Ca 2+ leaks through the Ca 2+ -ATPase of sarcoplasmic reticulum vesicles. In the presence of a transmembrane Ca 2+ concentration gradient, agents that modify the amount of ATP synthesized from ADP and P ; also modify the amount of heat produced by the hydrolysis of each ATP molecule. Thus, in the presence of heparin, less ATP is synthesized and more heat is produced. Conversely, with dimethyl sulfoxide more ATP is synthesized and less heat is produced. The data indicate that between limits (-10 to -30 kcal/mol) the Ca 2+ -ATPase can regulate the interconversion of energy in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is converted into other forms of energy.
The rates of hydrolysis of acetyl phosphate in the presence of 0.1 M NaOH and of ATP in the presence of either 1 M HCl or 1 M NaOH were measured at different temperatures and in the presence of different concentrations of the organic solvents dimethyl sulfoxide or ethylene glycol. Under all conditions tested, there was a progressive increase in the rate constant of hydrolysis of both phosphate compounds as the water activity of the medium was decreased by the addition of organic solvents. At 25"C, substitution of 70% of the water of the medium by dimethyl sulfoxide promoted an increase of two orders of magnitude in the rate constant of acetyl phosphate hydrolysis. In the presence of 80% and 90% dimethyl sulfoxide the rate of acetyl phosphate hydrolysis increased by more than two orders of magnitude and was so fast that it could not be measured with the method used. The effect of organic solvents on the rate of ATP hydrolysis was less pronounced than that observed for acetyl phosphate hydrolysis. At 30°C substitution of 90% of water by an organic solvent promoted a 44fold increase of the rate of ATP hydrolysis. Acceleration of either acetyl phosphate or ATP hydrolysis rates was promoted by a decrease in both activation energies (I&) and in entropies of activation d5'$. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the CaZ+-ATPase of sarcoplasmic reticulum and the F,-ATPase of mitochondria.
The first step towards ATP synthesis by the Ca2‐ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35°C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10‐fold, whereas it had no effect on the apparent affinity for Mg2+.
In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations.
Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+.
The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.