Mechanism for 3,3',4',5-tetrachlorosalicylanilide-induced activation of sarcoplasmic reticulum ATPase

Effects of 3,3',4',5-tetrachlorosalicylanilide (TCS), a lipophilic weak acid, on Ca2+ uptake and ATP hydrolysis by the sarcoplasmic reticulum calcium pump were characterized to obtain insight into the possible role of hydrophobic portions of the Ca2+-ATPase in the catalytic mechanism of the enzyme. TCS exhibited both the stimulatory and inhibitory effects on the calcium pump activities depending on its concentration. At optimal concentrations, it increased these activities by up to 5-fold at pH 7.0 and 6 degrees C. Analysis of partial reactions of ATP hydrolysis by the purified ATPase revealed that TCS accelerated Ca2+ release from the ADP-sensitive phosphoenzyme up to 6-fold, whereas it affected other reaction steps to a much less extent, indicating that the site of the stimulatory action of TCS is rather specific in terms of the reaction sequence. These effects of TCS became less prominent at higher temperatures, although the enzyme-TCS interactions as detected in the direct binding experiment or by measurement of quenching of protein fluorescence were not affected by a similar change in temperature. The TCS effects were also dependent on pH of the 8.0 suggested that the protonated form of TCS is responsible for both the stimulatory and inhibitory effects of the drug. These results, taken together with those obtained previously with a spin-labeled probe (Barratt, M. D., and Weaver, A. C. (1979) Biochim. Biophys. Acta 555, 337-348), may suggest that TCS stimulates the calcium pump activity through its effect on the lipid bilayer, although its direct action on hydrophobic portion(s) of the ATPase protein cannot be ruled out.