Effect of solubilization on adenosine 5'-triphosphate induced calcium release from purified sarcoplasmic reticulum calcium adenosinetriphosphatase.
ATP-induced Ca2+ release from the purified sarcoplasmic reticulum Ca2+-ATPase has been monitored in several different ATPase environments. Arsenazo III was used as a Ca2+ indicator in stopped-flow experiments and was shown to detect the early burst in Ca2+ transport, slower steady-state transport, and release of Ca2+ from fragmented sarcoplasmic reticulum. ATP-induced rapid release of Ca2+ followed by a slower rebinding step could be demonstrated for purified Ca2+-ATPase in leaky vesicles if the reaction was slowed by lowering the pH to 6.1 and by including dimethyl sulfoxide in the reaction medium. At a dodecyl octaoxyethylene glycol monoether (C12E8) to protein weight ratio of 0.2, a detergent concentration too low for solubilization to occur, ATP-induced Ca2+ release occurred more rapidly than for native leaky membranes, whereas the rebinding step was slower. In contrast, no Ca2+ release was observed for any soluble preparation. The kinetics of Ca2+ release was studied under conditions where the ATPase was monomeric or aggregated, and also in the presence of added phospholipid. The ATPase was shown to be monomeric by sedimentation equilibrium measurements in the presence of Ca2+, ADP, and beta, gamma-methylene-ATP at a C12E8 to protein weight ratio of 2.0. It is concluded that solubilization of the Ca2+-ATPase may result in uncoupling of ATP hydrolysis from ATP-induced Ca2+ release.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001150:Arsenazo III; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D046911:Macromolecular Substances; D001150:Arsenazo III; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide