Stoichiometry of tight binding of magnesium and fluoride to phosphorylation and high-affinity binding of ATP, vanadate, and calcium in the sarcoplasmic reticulum Ca(2+)-ATPase.
We previously showed that, when the purified Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, Asahikawa Medical College.
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Daiho
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Kubota
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Kanazawa
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005459:Fluorides / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates / Q000378:metabolism*
D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides; D002118:Calcium