Fluorometric titration of the sarcoplasmic reticulum adenosinetriphosphatase calcium sites in the presence of vanadate

Abstract

Titration of the specific calcium binding sites of sarcoplasmic reticulum ATPase was carried out by measurements of intrinsic fluorescence in the absence and in the presence of vanadate. The previous finding that vanadate binding to the enzyme inhibits high-affinity calcium binding was confirmed. In addition, taking advantage of the slow kinetics of vanadate association and dissociation from the enzyme, we were able to titrate the fraction of sites remaining in the high affinity state in the presence of non-saturating vanadate. These sites were demonstrated to retain the characteristics displayed by the high-affinity sites in the absence of vanadate, and yielded information consistent with a competitive inhibition between vanadate and calcium. Reversal of the vanadate effect and reconversion of the binding sites to the high-affinity state was demonstrated by adding appropriate calcium concentrations to the enzyme-vanadate complex, and showing the appearance of the intrinsic fluorescence signal which is indicative of calcium occupancy of the sites in the high-affinity state. Partial or total reversal of the vanadate effect was obtained with very slow kinetics following addition of micromolar calcium or, at a somewhat faster rate, following addition of millimolar calcium. The latter experiments yielded titration of the binding sites in the low-affinity state, with a dissociation constant of approx. 2 mM at neutral pH and 10 mM Mg 2+. The time course of the fluorescence rise following addition of calcium in the presence of vanadate was more rapid in ‘leaky’ than in native sarcoplasmic reticulum vesicles, suggesting an intravesicular orientation of the low-affinity calcium sites involved in the reversal of the vanadate effect. Our observations provide experimental support for the postulated mechanism of high- and low-affinity interconversion of the ATPase calcium binding sites, and its dependence on the occupancy of the phosphorylation site by vanadate.