Kinetic and thermodynamic control of ATP synthesis by sarcoplasmic reticulum adenosinetriphosphatase.
Several experimental parameters, critical to the analysis of ATP synthesis by sarcoplasmic reticulum ATPase, were determined experimentally. 1) The phosphorylated enzyme intermediate obtained with acetylphosphate in the presence of a Ca2+ gradient was shown to be entirely ADP sensitive but quite stable in the absence of added ADP. On the contrary, the phosphoenzyme obtained with ATP is unstable due to the ADP formed during the phosphoryl transfer reaction. For this reason, addition of ADP to [32P]phosphoenzyme obtained with [32P]acetylphosphate provides the simplest conditions for kinetic studies on [gamma-32P]ATP synthesis. 2) The dissociation rate constant of newly synthesized ATP (in the reverse direction of the ATPase cycle) was measured experimentally and found to be 16 s-1. This value agrees well with the dissociation rate constant determined for adenyl-5'-yl imidodiphosphate bound to this enzyme. 3) ATP synthesis observed in the absence of a Ca2+ gradient was shown to be a kinetic overshoot due to ligand-induced perturbation of a limited number of partial reactions and occurring before equilibration of the entire system. Most of the ATP formed under these conditions was subsequently hydrolyzed as the overall equilibrium was reached. 4) Based on these and other (previously characterized) parameters, satisfactory simulations of single and multiple cycle ATP synthesis, in the presence and in the absence of a Ca2+ gradient, were obtained.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D002122:Calcium Chloride / Q000378:metabolism / Q000494:pharmacology; D002132:Calcium Radioisotopes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013816:Thermodynamics
D002132:Calcium Radioisotopes; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002122:Calcium Chloride