Control of energy fluxes by the sarcoplasmic reticulum Ca2+-ATPase: ATP hydrolysis, ATP synthesis and heat production.

The experiments described indicate that heat is released when Ca2+ leaks through the Ca2+-ATPase of sarcoplasmic reticulum vesicles. In the presence of a transmembrane Ca2+ concentration gradient, agents that modify the amount of ATP synthesized from ADP and Pi 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 Ca2+-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.

Uncoupling of muscle and blood platelets Ca2+ transport ATPases by heparin. Regulation by K+.

Heparin (1-10 micrograms/ml) inhibits the Ca2+ transport ATPases found in the sarcoplasmic reticulum of skeletal muscle, the plasma membrane of red cells, and the dense tubular system of blood platelets, but not the (Na+ + K+)-ATPase or the mitochondrial F1-ATPase. In the reversal of the Ca2+ pump, heparin uncouples the synthesis of ATP from Ca2+ efflux and inhibits the phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase by Pi, but has no effect the phosphorylation by ATP. The effect of heparin on the muscle Ca(2+)-ATPase is abolished by KCl and NaCl (100 mM) and to a lesser extent by LiCl. These monovalent cations are not effective as antagonists of heparin on the platelet Ca(2+)-ATPase. The effects of heparin are antagonized by the polyamines spermine, spermidine, and ruthenium red. Unlike KCl, polyamines are equally effective in counteracting the effects of heparin in both muscle and platelet Ca(2+)-ATPases. In addition to heparin, the Ca(2+)-ATPases of muscle and blood platelets are also inhibited by dextran sulfate and fucose-branched chondroitin sulfate. The inhibition promoted by these glycosaminoglycans is antagonized by monovalent cations and polyamines in the same manner as heparin. Heparan sulfate, chondroitin sulfate, and hyaluronic acid have not effect on the Ca(2+)-ATPases studied.

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

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 degrees 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.

Functional interactions of catalytic site and transmembrane channel in the sarcoplasmic reticulum ATPase.

Vesicular fragments of longitudinal sarcoplasmic reticulum were loaded with calcium by active transport, sedimented by centrifugation, and resuspended in neutral buffer and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Under these conditions, calcium efflux from the loaded vesicles occurred at rates varying from 100 to 700 nmol/mg/min, depending on the calcium load. If either Ca2+ (microM), Mg2+ (mM), K+ or Na+ (greater than 10 mM) were added to the resuspension medium, the rate of efflux was reduced. In the presence of Mg2+ and EGTA, a large inhibition of calcium efflux was produced by formation of phosphoenzyme intermediate with Pi. In this case, addition of ADP again started calcium efflux, coupled with ATP synthesis. The rates of uncoupled or coupled efflux were approximately the same. The observed calcium fluxes are attributed to a slow channel formed by ATPase transmembrane helices (MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 686-700) and are capable of long range interaction with the catalytic site. Coupling of transport and catalytic activities is thereby produced by phosphorylation and ligand binding. The channel includes negatively charged residues that are likely to influence calcium fluxes through cation binding. It is proposed that this channel is the mechanistic device for active transport of calcium across the sarcoplasmic reticulum membrane, and for its reversal.

Role of water activity on the rates of acetyl phosphate and ATP hydrolysis.

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 degrees 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 degrees C, substitution of 90% of water by an organic solvent promoted a 4-6-fold 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 (Ea) and in entropies of activation delta S. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the Ca2+-ATPase of sarcoplasmic reticulum and the F1-ATPase of mitochondria.