title: Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation.
abstract: The mechanism of sarcoplasmic reticulum (SR) ATPase Mg2+-dependent phosphorylation from Pi was investigated in the presence of 15% v/v dimethyl sulfoxide at pH 6, 20 degrees C, and in the absence of potassium. Measurements of intrinsic fluorescence changes and of 32P-labeled phosphoprotein (*E-P) were in agreement, both at equilibrium and in transient situations. We found that the amount of phosphoenzyme present and its rate of formation depended solely on the concentration of the (Mg X Pi) complex. Up to 6 nmol of phosphate/mg of protein was covalently bound to the enzyme, implying almost complete phosphorylation. Oxygen exchange experiments were also performed in order to allow calculation of the absolute rate constant of *E-P hydrolysis to the noncovalent complex (0.8-1.0 s-1), which differs from the observed rate of enzyme dephosphorylation (0.3-0.5 s-1); in addition, they allowed calculation of the bimolecular rate constant of substrate binding (2-2.4 M-1 s-1). The results demonstrate that in the presence of dimethyl sulfoxide, phosphorylation occurs by the following simple mechanism: relatively slow binding of the neutral substrate (Mg X Pi), with poor affinity, followed by a thermodynamically favorable formation of the covalent bond between phosphate and the possibly hydrophobic active site. The interaction between magnesium and calcium-deprived SR vesicles was studied in the presence of 0-20% v/v dimethyl sulfoxide (or 0-30% v/v glycerol) at pH 7 and 20 degrees C. The presence of either solvent led to the disappearance of the two typical pH-dependent effects we previously characterized for magnesium: loss of the Mg2+-induced spectral shift of tryptophan fluorescence emission and loss of the biphasic pattern displayed by the intrinsic fluorescence rise after addition of calcium to Ca2+-deprived Mg2+-preincubated vesicles. In the absence of solvent, the interaction of magnesium with the calcium-deprived ATPase was also characterized from the point of view of phosphoenzyme formation from ATP or Pi at pH 7 in the absence of potassium: we found that calcium-independent phosphorylation was slower when phosphate was added to SR vesicles preincubated with magnesium that when magnesium was added to vesicles preincubated with phosphate, suggesting that preincubation with magnesium had depleted the phosphate-reactive conformation of the ATPase. A simple reaction scheme for phosphoenzyme formation is described: it implies that the (Mg X Pi) complex is a substrate for this reaction, whereas the Mg2+ itself acts as a pH-dependent, dimethyl sulfoxide sensitive inhibitor of full enzyme phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.forename: P
authors.initials: P
authors.lastname: Champeil
authors.forename: F
authors.initials: F
authors.lastname: Guillain
authors.forename: C
authors.initials: C
authors.lastname: Vénien
authors.forename: M P
authors.initials: MP
authors.lastname: Gingold
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012997:Solvents; D013050:Spectrometry, Fluorescence
chemical_list: D010103:Oxygen Isotopes; D010710:Phosphates; D012997:Solvents; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium; D002118:Calcium; D004121:Dimethyl Sulfoxide