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+ cooperativity (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca 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 Ca, calculated from medium Pi ⇌ 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 Ca 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 ⇌ ATP exchange was inhibited 65%. Solubilization of the vesicles [0.2% (w/v) Triton X-100 and 5 mg of Triton X-100/mg of protein] completely or partially reversed these changes except for the K0.5(Ca) and nH values for ATPase activity, the rate of p-nitrophenyl phosphate hydrolysis at saturating Ca2+ concentrations, and the phosphoenzyme levels formed from Pi. It is proposed that intercalation of Triton X-100 into sarcoplasmic reticulum vesicles interacts with the ATPase, producing a functionally modified enzyme with low affinity for Ca2+ and no cooperativity between Ca2+ binding sites and which is partially inhibited in several catalytic properties. The inhibition, at least in part, is accounted for by the lower rate constant for phosphoenzyme hydrolysis. A large increase in the fluidity of the environment of the ATPase in the detergent micelle may account for the restoration of the Ca2+-induced conformational change and catalytic properties of the solubilized ATPase. The relatively large increase in K0.5(Ca) for ATPase activity following solubilization can be largely ascribed to a loss of ATP enhancement of Ca2+ binding affinity. © 1984, American Chemical Society. All rights reserved.