Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on calcium(2+) binding and calcium(2+)-ATPase activity of sarcoplasmic reticulum

Abstract

The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of [rabbit] sarcoplasmic reticulum was investigated. The changes observed were compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% wt/vol 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 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 .apprx. 1/2 that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi .dblarw. 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 by .apprx. 1/2 at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi .dblarw. ATP exchange was inhibited 65%. Solubilization of the vesicles [0.2% (wt/vol) 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. Intercalation of Triton X-100 into sarcoplasmic reticulum vesicles apparently 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 lowr 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+ bindng affinity.