Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on Ca2+ binding and Ca2+-ATPase activity of sarcoplasmic reticulum.
The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of sarcoplasmic reticulum has been investigated. The changes observed have been compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% (w/v) Triton X-100, concentrations which did not solubilize the vesicles but completely inhibit ATP-mediated Ca2+ accumulation, 8-16 mol of detergent/mol of ATPase was associated with the vesicles. This amount of Triton X-100 altered equilibrium Ca2+ binding and Ca2+ activation of p-nitrophenyl phosphate and of ATP hydrolysis in a manner which lowered the apparent Ca2+ cooperatively (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca2+ binding and activation parameters were associated with a 90% lower Ca2+-induced change in fluorescence of fluorescein isothiocyanate modified enzyme. The rates of p-nitrophenyl phosphate and of ATP hydrolysis, at saturating Ca2+ concentrations, were about half that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi in equilibrium HOH exchange and phosphoenzyme measurements, was lowered from 38 to 11 s-1. The steady-state level of phosphoenzyme formed from Pi in the absence of Ca2+ was slightly increased up to 0.02% Triton X-100 and then decreased about half at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi in equilibrium ATP exchange was inhibited 65%.(ABSTRACT TRUNCATED AT 250 WORDS)
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D012995:Solubility
D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D009597:4-Nitrophenylphosphatase; D000252:Calcium-Transporting ATPases; D002118:Calcium