Calorimetric studies of the mechanism of the Ca2+-ATPase reaction of sarcoplasmic reticulum.
The ATP hydrolysis catalyzed by sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was studied at 6 degrees C by microcalorimetry in parallel with measuring the extent of hydrolysis (xi ATP) and the level of the phosphorylated intermediate (E approximately P) of the Ca2+-ATPase (xi EP) in the reaction medium. In order to assign whether the formation or decomposition of E approximately P is responsible for the liberation of protons upon ATP hydrolysis, buffers with different heats of protonation (delta H buffer) were used. They were 3-(N-morpholino)propanesulfonic acid (MOPS, delta H buffer = -21.0 kJ X mol-1) and 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP, delta H buffer = -43.4 kJ X mol-1). The apparent heats of the overall ATP hydrolysis coupled to Ca2+ uptake (delta HATP/Ca) in MOPS and BTP were -36.2 and -61.1 kJ X mol-1, respectively. These values together with the values of delta H buffer give an estimate of 1.1 mol of protons released on hydrolysis of 1 mol of ATP coupled to Ca2+ uptake. This H+/ATP ratio is in good agreement with that determined by pH measurement (Kodama, T., Kurebayashi, N., & Ogawa, Y. (1980) J. Biochem. 88, 1259-1265). From the values of observed heat, xi ATP, xi EP, and delta HATP/Ca in MOPS-buffered medium, the heat of E approximately P formation was calculated to be + 56 kJ X mol-1, which was very similar to that obtained for BTP-buffered medium.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002021:Buffers; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002151:Calorimetry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D002021:Buffers; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases