Studies on Structure and Function of the ATP-Binding sites of the Sarcoplasmic Reticulum Calcium Pump.

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In an attempt to establish the stoichiometry of the phosphorylatable catalytic site to the specific fluorescein 5-isothiocyanate (FITC) binding site (Lys-515) in the Ca^<2+>-ATPase of sarcoplasmic reticulum vescicles (SRV), labeling by FITC or phosphorylation by ATP (or Pi) was : performed with SRV under the conditions in which almost all of the specific FITC binding sites or phosphorylatable catalytic sites can be labeled or phosphorylated. The resultant SRV were solubilized in lithium dodecyl sulfate, and then the Ca^<2+>ATPase was purified by using a size exclusion high performance liquid chromatography. The contents of bound FITC and phosphoenzyme in the ATPase isolated as above were determined. Peptide maapping of the tryptic digests and sequencing showed that Lys-515 of the Ca^<2+>-ATPase was exclusively labeled with FITC.This specific labeling was completely prevented by ATP.The content of phosphoenzyme (4.57 and 4.94 nmol/mg of protein from ATP and Pi respectively) was approximately half that of the specific FITC binding site (8.16-8.19 nmol/m of protein) and also half that of the llO-KDa Ca^<2+>ATPase chain (9.06 nmol/mg of protein) calculated on the assumption that the isolated ATPase chain was pure. These findings are consistent with a possible half-of-the-sites reactivity, being in favor of a dimeric structure of the Ca^<2+>ATPase in SRV.The extent of the specific FITC binding required for complete inhibition of ATP-induced phosphorylation corresponded to 6.4 nmol of bound FITC/mg of protein. This finding suggests that FITC somewhat preferentially binds to one half of the ATPase chains in SRV and that this binding is primarily responsible for the observed inhibition of phosphorylation.