STOICHIOMETRY OF TIGHT-BINDING OF MAGNESIUM AND FLUORIDE TO PHOSPHORYLATION AND HIGH-AFFINITY BINDING OF ATP, VANADATE, AND CALCIUM IN THE SARCOPLASMIC-RETICULUM CA2+-ATPASE

We previously showed that, when the purified Ca2+-ATPase from sarcoplasmic reticulum (SR) is treated with fluoride (F-) in the presence of Mg2+, a complete inactivation of the enzyme is induced by tight binding of approximately 2 mol of Mg2+ and 4 mol of F- to the catalytic site per mole of phosphorylation site [Kubota, T., Daiho, T., & Kanazawa, T. (1993) Biochim. Biophys. Acta 1163, 131-143]. Contradictorily, on the basis of the postulated content of the Ca2+-ATPase in F--treated SR vesicles, Coll and Murphy [(1992) J. Biol. Chem. 267, 21584-21587] suggested that each inactivated enzyme contains one tightly-bound Mg2+ and two tightly-bound F-. The present study has been made to resolve this conflict. The contents of phosphorylation site, high-affinity ATP-binding site, high-affinity vanadate-binding site, and high-affinity Ca2+-binding site in the SR vesicles used were 3.33 +/- 0.06, 3.54 +/- 0.12, 3.34 +/- 0.04, and 6.98 +/- 0.16 nmol/mg, respectively. When the vesicles were incubated with F- in the presence of Mg2+, the Ca2+-ATPase was inactivated progressively. After removal of unbound Mg2+ and F- by gel filtration, tightly-bound Mg2+ and F- were determined by use of an atomic absorption spectrophotometer and a F--selective electrode. A linear relationship existed between the extent of the enzyme inactivation and the contents of the tightly-bound ligands. The contents of tightly-bound Mg2+ and F- in the fully inactivated vesicles were 6.65 and 12.6 nmol/mg, respectively. The same stoichiometry was obtained with another preparation of SR vesicles. These results demonstrate that tight binding of 2 mol of Mg2+ and 4 mol of F- per mole of phosphorylation site, per mole of high-affinity ATP- or vanadate-binding site, and per 2 mol of high-affinity Ca2+-binding site leads to a complete inactivation of the Ca2+-ATPase in SR vesicles.