Negative or Positive Cooperation in Calcium Binding to Detergent-solubilized ATPase of the Sarcoplasmic Reticulum

Two different conformations of chemically equivalent Ca2+-ATPase molecules in the sarcoplasmic reticulum have been shown to non- and positive cooperatively bind two calcium ions, respectively (Nakamura, J.(1994) J. Biol. Chem. 269, 30822-30827). At pH 7.40, these ATPase molecules split into E1 (high affinity state for calcium) and E2 (low affinity state for calcium), respectively, before calcium binding. At this pH, calcium binding to the monomeric ATPase, solubilized with dodecyloctaethylenglycol monoether, was studied by examining 45Ca2+ binding to the ATPase and calcium dependences of its phosphorylation, fluorescence intensity, ATP-hydrolysis at of and the solubilized ATPase molecules in E2 and cooperatively bind of of the ATPase with calcium affinity of of calcium at the ATPase molecules to the of the of the binding and the to Two different conformations of chemically equivalent Ca2+-ATPase molecules in the sarcoplasmic reticulum have been shown to non- and positive cooperatively bind two calcium ions, respectively (Nakamura, J.(1994) J. Biol. Chem. 269, 30822-30827). At pH 7.40, these ATPase molecules split into E1 (high affinity state for calcium) and E2 (low affinity state for calcium), respectively, before calcium binding. At this pH, calcium binding to the monomeric ATPase, solubilized with dodecyloctaethylenglycol monoether, was studied by examining 45Ca2+ binding to the ATPase and calcium dependences of its phosphorylation, fluorescence intensity, ATP-hydrolysis at of and the solubilized ATPase molecules in E2 and cooperatively bind of of the ATPase with calcium affinity of of calcium at the ATPase molecules to the of the of the binding and the to reticulum Ca2+-ATPase calcium of calcium the sarcoplasmic reticulum by with of of the Ca2+-ATPase molecules in in the J. Biol. Chem. J. Biol. monomeric been shown to the of of the and to of the calcium J. Biol. Chem. J. Biol. Chem. J. Biol. Chem. the two different conformations of chemically equivalent ATPase molecules in the J. J. Biol. Chem. J. J. Biol. Chem. of in E1 (high affinity state for calcium) and E2 (low affinity state for calcium) before calcium binding and two calcium with affinity for the calcium in E2 of pH before calcium binding and positive cooperatively two calcium with affinity for the calcium of the chemically equivalent ATPase molecules these of the ATPase molecules in the the of to of the of the molecules in the by of the binding at the monomeric been studied for the by the solubilized with been to for the of the J. Biol. Chem. J. Biol. Chem. of calcium of by the J. Biol. Chem. and J. Biol. Chem. this positive cooperatively calcium with the of the in the of the been for in fluorescence of the J. Biol. Chem. and J. Biol. Chem. of the by of the the of calcium binding at the ATPase have shown the ATPase of the solubilized in the of calcium and J. Biol. Chem. fluorescence of the to the of the J. Biol. Chem. monomeric for the calcium binding to the solubilized was by the of calcium binding to the solubilized calcium binding to the and calcium dependences of its phosphorylation, fluorescence intensity, at of and the of the solubilized E1 and E2 and calcium binding of the with of the at pH the two of shown to split into E1 and respectively, before calcium binding and to and bind calcium in non- and positive the of the by of the solubilized molecules in E2 and in calcium binding. of the binding was at of for of the sarcoplasmic reticulum of the in J. Ca2+-ATPase was the sarcoplasmic reticulum by the sarcoplasmic reticulum with in the J. for in the of to the reticulum to was by the J. Biol. Chem. with the of the of the with of the ATPase was to the of was solubilized with to the of and the was by At the the ATPase was at of of the was at of of the of the was for in the solubilized to this of the ATPase by of the the of the the ATPase was solubilized in of the with of the and the to the molecules J. Biol. Chem. J. Biol. Chem. for to of the to the in the of to solubilized by the by the of of the to the of the fluorescence of the before and of the at for ATPase of the solubilized ATPase was of of in of and at pH and of the was of in and at pH and calcium binding to the solubilized ATPase was to the of J. Biol. Chem. of was solubilized in and was for of calcium binding at of the calcium of was of and of and of was at of was in of and by the at the At the the was by the of of with of of the was was by the of of with of and of the at the was at the the of the was was the of the and the the was of the was and the of the was the of the and the of to to two of the this of the of the in this was by the was in J. J. Biol. Chem. was in and for at and of to the At the to of the ATPase of the ATPase at of by At the in the and of At was was to the was for was J. in fluorescence of the was by the and for and respectively, in J. J. Biol. Chem. the of the solubilized in the of calcium and of for of the ATPase before the was of the solubilized at was at by the in of of the and at the of to the the was in the for was At of the of was in and for at the the was by the of was was by the of at of the with and of the was in of of the and at was in the for before of the by the of was of was to the of and J. Biol. Chem. of Ca2+-ATPase in the sarcoplasmic reticulum chemically equivalent have been shown to in E1 and E2 and of pH in respectively, before calcium binding at J. J. Biol. Chem. J. J. Biol. Chem. and bind two calcium ions, respectively, pH and of pH, and and positive cooperatively in the calcium At pH 7.40, the two of Ca2+-ATPase in the have been shown to split into E1 and respectively, and and bind J. J. Biol. Chem. J. J. Biol. Chem. the to calcium binding to the monomeric with to the at this shown in to the calcium binding to the solubilized of the was at was by the of calcium to the was with was at of in of the two was in E1 before calcium binding J. J. Biol. Chem. was of the solubilized molecules in E2 before calcium binding and bind calcium of the of the E2 to E1 the of in fluorescence of the of calcium the with of the the fluorescence at was to the at been to the solubilized in the of J. Biol. Chem. calcium of the fluorescence in the calcium with of and calcium affinity of was at was the at solubilized to in calcium binding of and of the at of and the in with of the fluorescence been shown to for the the of in calcium binding of the solubilized was At the of calcium to the solubilized was of At this calcium of calcium binding of the to with calcium the of the fluorescence intensity, the and the shown in of the was of the solubilized two calcium of the for of the with the in the by J. Biol. Chem. the solubilized cooperatively two calcium ions, calcium binding at of the by in calcium affinity at and binding to monomeric ATPase to in state calcium with the two of ATPase molecules in the and positive cooperatively bind two calcium ions, J. J. Biol. Chem. the of the molecules for E1 and E2 and for of the binding by of the of the ATPase molecules two different conformations of the of of the monomeric the monomeric in state of and into two different conformations of the in the this calcium binding at the monomeric with of the ATPase of calcium calcium of the fluorescence of was in of and of to the calcium and of the fluorescence the of and respectively, of the was of the of the calcium the of the at calcium to the of the of calcium at of ATPase was by the was in the of in of for was calcium of the ATPase of the the of the at calcium to the of of of the of of calcium calcium of the of the the of the at calcium to the of of of the with the of the of calcium of the solubilized at of calcium of of the solubilized been shown to have positive with J. Biol. Chem. J. Biol. Chem. of calcium was at in J. Biol. Chem. J. Biol. Chem. the of the was with was was at the of the of the monomeric in the of the binding of the to positive and in the in the affinity for the monomeric to state to calcium with the of the the of by was shown in the by of to binding at the of the the binding at the to at binding the of the of the with affinity J. J. Biol. Chem. J. Biol. Chem. for J. at the in the solubilized with and with have been J. Biol. Chem. in the with the of the calcium of the been shown to have the of J. Biol. Chem. this the of the by of been shown to of of two in the the and the of in and to E2 and and the the of the E2 to J. Biol. Chem. been to the been to the J. the the of the to in calcium the of calcium of the fluorescence was the was the in calcium binding of the in the of the E2 to of calcium at of calcium of the ATPase of the the of the at calcium to the of of of the reticulum Ca2+-ATPase calcium of calcium the sarcoplasmic reticulum by with of of the Ca2+-ATPase molecules in in the J. Biol. Chem. J. Biol. monomeric been shown to the of of the and to of the calcium J. Biol. Chem. J. Biol. Chem. J. Biol. Chem. the two different conformations of chemically equivalent ATPase molecules in the J. J. Biol. Chem. J. J. Biol. Chem. of in E1 (high affinity state for calcium) and E2 (low affinity state for calcium) before calcium binding and two calcium with affinity for the calcium in E2 of pH before calcium binding and positive cooperatively two calcium with affinity for the calcium of the chemically equivalent ATPase molecules these of the ATPase molecules in the the of to of the of the molecules in the by of the binding at the monomeric been studied for the by the solubilized with been to for the of the J. Biol. Chem. J. Biol. Chem. of calcium of by the J. Biol. Chem. and J. Biol. Chem. this positive cooperatively calcium with the of the in the of the been for in fluorescence of the J. Biol. Chem. and J. Biol. Chem. of the by of the the of calcium binding at the ATPase have shown the ATPase of the solubilized in the of calcium and J. Biol. Chem. fluorescence of the to the of the J. Biol. Chem. monomeric for the calcium binding to the solubilized was by the of calcium binding to the solubilized calcium binding to the and calcium dependences of its phosphorylation, fluorescence intensity, at of and the of the solubilized E1 and E2 and calcium binding of the with of the at pH the two of shown to split into E1 and respectively, before calcium binding and to and bind calcium in non- and positive the of the by of the solubilized molecules in E2 and in calcium binding. of the binding was at of for of the sarcoplasmic reticulum of the in J. Ca2+-ATPase was the sarcoplasmic reticulum by the sarcoplasmic reticulum with in the J. for in the of to the reticulum to was by the J. Biol. Chem. with the of the of the with of the ATPase was to the of was solubilized with to the of and the was by At the the ATPase was at of of the was at of of the of the was for in the solubilized to this of the ATPase by of the the of the the ATPase was solubilized in of the with of the and the to the molecules J. Biol. Chem. J. Biol. Chem. for to of the to the in the of to solubilized by the by the of of the to the of the fluorescence of the before and of the at for ATPase of the solubilized ATPase was of of in of and at pH and of the was of in and at pH and calcium binding to the solubilized ATPase was to the of J. Biol. Chem. of was solubilized in and was for of calcium binding at of the calcium of was of and of and of was at of was in of and by the at the At the the was by the of of with of of the was was by the of of with of and of the at the was at the the of the was was the of the and the the was of the was and the of the was the of the and the of to to two of the this of the of the in this was by the was in J. J. Biol. Chem. was in and for at and of to the At the to of the ATPase of the ATPase at of by At the in the and of At was was to the was for was J. in fluorescence of the was by the and for and respectively, in J. J. Biol. Chem. the of the solubilized in the of calcium and of for of the ATPase before the was of the solubilized at was at by the in of of the and at the of to the the was in the for was At of the of was in and for at the the was by the of was was by the of at of the with and of the was in of of the and at was in the for before of the by the of was of was to the of and J. Biol. Chem. for of the sarcoplasmic reticulum of the in J. Ca2+-ATPase was the sarcoplasmic reticulum by the sarcoplasmic reticulum with in the J. for in the of to the reticulum to was by the J. Biol. Chem. with the of the of the with of the ATPase was to the of was solubilized with to the of and the was by At the the ATPase was at of of the was at of of the of the was for in the solubilized to this of the ATPase by of the the of the the ATPase was solubilized in of the with of the and the to the molecules J. Biol. Chem. J. Biol. Chem. for to of the to the in the of to solubilized by the by the of of the to the of the fluorescence of the before and of the at for ATPase of the solubilized ATPase was of of in of and at pH and of the was of in and at pH and calcium binding to the solubilized ATPase was to the of J. Biol. Chem. of was solubilized in and was for of calcium binding at of the calcium of was of and of and of was at of was in of and by the at the At the the was by the of of with of of the was was by the of of with of and of the at the was at the the of the was was the of the and the the was of the was and the of the was the of the and the of to to two of the this of the of the in this was by the was in J. J. Biol. Chem. was in and for at and of to the At the to of the ATPase of the ATPase at of by At the in the and of At was was to the was for was J. in fluorescence of the was by the and for and respectively, in J. J. Biol. Chem. the of the solubilized in the of calcium and of for of the ATPase before the was of the solubilized at was at by the in of of the and at the of to the the was in the for was At of the of was in and for at the the was by the of was was by the of at of the with and of the was in of of the and at was in the for before of the by the of was of was to the of and J. Biol. Chem. for of the sarcoplasmic reticulum of the in J. Ca2+-ATPase was the sarcoplasmic reticulum by the sarcoplasmic reticulum with in the J. for in the of to the reticulum to was by the J. Biol. Chem. with the of the of the with of the ATPase was to the of was solubilized with to the of and the was by At the the ATPase was at of of the was at of of the of the was for in the solubilized to this of the ATPase by of the the of the the ATPase was solubilized in of the with of the and the to the molecules J. Biol. Chem. J. Biol. Chem. for to of the to the in the of to solubilized by the by the of of the to the of the fluorescence of the before and of the at for ATPase of the solubilized ATPase was of of in of and at pH and of the was of in and at pH and calcium binding to the solubilized ATPase was to the of J. Biol. Chem. of was solubilized in and was for of calcium binding at of the calcium of was of and of and of for of the sarcoplasmic reticulum of the in J. Ca2+-ATPase was the sarcoplasmic reticulum by the sarcoplasmic reticulum with in the J. for in the of to the reticulum to was by the J. Biol. Chem. with the of the of the with of the ATPase was to the of was solubilized with to the of and the was by At the the ATPase was at of of the was at of of the of the was for in the solubilized to this of the ATPase by of the the of the the ATPase was solubilized in of the with of the and the to the molecules J. Biol. Chem. J. Biol. Chem. for to of the to the in the of to solubilized by the by the of of the to the of the fluorescence of the before and of the at for ATPase of the solubilized ATPase was of of in of and at pH and of the was of in and at pH and calcium binding to the solubilized ATPase was to the of J. Biol. Chem. of was solubilized in and was for of calcium binding at of the calcium of was of and of and of was at of was in of and by the at the At the the was by the of of with of of the was was by the of of with of and of the at the was at the the of the was was the of the and the the was of the was and the of the was the of the and the of to to two of the this of the of the in this was by the was in J. J. Biol. Chem. was at of was in of and by the at the At the the was by the of of with of of the was was by the of of with of and of the at the was at the the of the was was the of the and the the was of the was and the of the was the of the and the of to to two of the this of the of the in this was by the was in J. J. Biol. Chem. was in and for at and of to the At the to of the ATPase of the ATPase at of by At the in the and of At was was to the was for was J. in fluorescence of the was by the and for and respectively, in J. J. Biol. Chem. was in and for at and of to the At the to of the ATPase of the ATPase at of by At the in the and of At was was to the was for was J. in fluorescence of the was by the and for and respectively, in J. J. Biol. Chem. the of the solubilized in the of calcium and of for of the ATPase before the was of the solubilized at was at by the in of of the and at the of to the the was in the for was At of the of was in and for at the the was by the of was was by the of at of the with and of the was in of of the and at was in the for before of the by the of was of was to the of and J. Biol. Chem. the of the solubilized in the of calcium and of for of the ATPase before the was of the solubilized at was at by the in of of the and at the of to the the was in the for was At of the of was in and for at the the was by the of was was by the of at of the with and of the was in of of the and at was in the for before of the by the of was of was to the of and J. Biol. Chem. of Ca2+-ATPase in the sarcoplasmic reticulum chemically equivalent have been shown to in E1 and E2 and of pH in respectively, before calcium binding at J. J. Biol. Chem. J. J. Biol. Chem. and bind two calcium ions, respectively, pH and of pH, and and positive cooperatively in the calcium At pH 7.40, the two of Ca2+-ATPase in the have been shown to split into E1 and respectively, and and bind J. J. Biol. Chem. J. J. Biol. Chem. the to calcium binding to the monomeric with to the at this shown in to the calcium binding to the solubilized of the was at was by the of calcium to the was with was at of in of the two was in E1 before calcium binding J. J. Biol. Chem. was of the solubilized molecules in E2 before calcium binding and bind calcium of the of the E2 to E1 the of in fluorescence of the of calcium the with of the the fluorescence at was to the at been to the solubilized in the of J. Biol. Chem. calcium of the fluorescence in the calcium with of and calcium affinity of was at was the at solubilized to in calcium binding of and of the at of and the in with of the fluorescence been shown to for the the of in calcium binding of the solubilized was At the of calcium to the solubilized was of At this calcium of calcium binding of the to with calcium the of the fluorescence intensity, the and the shown in of the was of the solubilized two calcium of the for of the with the in the by J. Biol. Chem. the solubilized cooperatively two calcium ions, calcium binding at of the by in calcium affinity at and binding to monomeric ATPase to in state calcium with the two of ATPase molecules in the and positive cooperatively bind two calcium ions, J. J. Biol. Chem. the of the molecules for E1 and E2 and for of the binding by of the of the ATPase molecules two different conformations of the of of the monomeric the monomeric in state of and into two different conformations of the in the this calcium binding at the monomeric with of calcium at of ATPase was by the was in the of in of for was calcium of the ATPase of the the of the at calcium to the of of of the of of calcium calcium of the of the the of the at calcium to the of of of the with the of the of calcium of the solubilized at of calcium of of the solubilized been shown to have positive with J. Biol. Chem. J. Biol. Chem. of calcium was at in J. Biol. Chem. J. Biol. Chem. the of the was with was was at the of the of the monomeric in the of the binding of the to positive and in the in the affinity for the monomeric to state to calcium with the of the the of by was shown in the by of to binding at the of the the binding at the to at binding the of the of the with affinity J. J. Biol. Chem. J. Biol. Chem. for J. at the in the solubilized with and with have been J. Biol. Chem. in the with the of the calcium of the been shown to have the of J. Biol. Chem. this the of the by of been shown to of of two in the the and the of in and to E2 and and the the of the E2 to J. Biol. Chem. been to the been to the J. the the of the to in calcium the of calcium of the fluorescence was the was the in calcium binding of the in the of the E2 to of calcium at of calcium of the ATPase of the the of the at calcium to the of of of the Two of Ca2+-ATPase in the sarcoplasmic reticulum chemically equivalent have been shown to in E1 and E2 and of pH in respectively, before calcium binding at J. J. Biol. Chem. J. J. Biol. Chem. and bind two calcium ions, respectively, pH and of pH, and and positive cooperatively in the calcium At pH 7.40, the two of Ca2+-ATPase in the have been shown to split into E1 and respectively, and and bind J. J. Biol. Chem. J. J. Biol. Chem. the to calcium binding to the monomeric with to the at this shown in to the calcium binding to the solubilized of the was at was by the of calcium to the was with was at of in of the two was in E1 before calcium binding J. J. Biol. Chem. was of the solubilized molecules in E2 before calcium binding and bind calcium of the of the E2 to E1 the of in fluorescence of the of calcium the with of the the fluorescence at was to the at been to the solubilized in the of J. Biol. Chem. calcium of the fluorescence in the calcium with of and calcium affinity of was at was the at solubilized to in calcium binding of and of the at of and the in with of the fluorescence been shown to for the the of in calcium binding of the solubilized was At the of calcium to the solubilized was of At this calcium of calcium binding of the to with calcium the of the fluorescence intensity, the and the shown in of the was of the solubilized two calcium of the for of the with the in the by J. Biol. Chem. the solubilized cooperatively two calcium ions, calcium binding at of the by in calcium affinity at and binding to monomeric ATPase to in state calcium with the two of ATPase molecules in the and positive cooperatively bind two calcium ions, J. J. Biol. Chem. the of the molecules for E1 and E2 and for of the binding by of the of the ATPase molecules two different conformations of the of of the monomeric the monomeric in state of and into two different conformations of the in the this calcium binding at the monomeric with with the of the of calcium of the solubilized at of calcium of of the solubilized been shown to have positive with J. Biol. Chem. J. Biol. Chem. of calcium was at in J. Biol. Chem. J. Biol. Chem. the of the was with was was at the of the of the monomeric in the of the binding of the to positive and in the in the affinity for the monomeric to state to calcium with the of the the of by was shown in the by of to binding at the of the the binding at the to at binding the of the of the with affinity J. J. Biol. Chem. J. Biol. Chem. for J. at the in the solubilized with and with have been J. Biol. Chem. in the with the of the calcium of the been shown to have the of J. Biol. Chem. this the of the by of been shown to of of two in the the and the of in and to E2 and and the the of the E2 to J. Biol. Chem. been to the been to the J. the the of the to in calcium the of calcium of the fluorescence was the was the in calcium binding of the in the of the E2 to of the of for to fluorescence