title: Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions.
abstract: We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000494:pharmacology*
chemical_list: D003902:Detergents; D013501:Surface-Active Agents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases