Interaction of Calcium and Vanadate with Fluorescein Isothiocyanate Labeled Ca²⁺-ATPase from Sarcoplasmic Reticulum: Kinetics and Equilibria

The interaction of Ca²⁺ and vanadate with fluorescein isothiocyanate (FITC) labeled sarcoplasmic reticulum (SR) Ca²⁺-ATPase has been studied by following the kinetics of changes in the reporter group fluorescence and equilibrium fluorescence levels. The vanadate species bound to the enzyme is clearly monomeric orthovanadate, probably H₂VO₄^-. Vanadate binding is noncooperative, suggesting an absence of interactions between the Ca²⁺-ATPase subunits. The fluorescence experiments confirm the existence of a calcium-enzyme-vanadate complex (in the presence of magnesium). On the basis of the fluorescence properties of this complex, it is similar in its conformation to the calcium-enzyme complex, i.e., “E₁-like” rather than “E₂-like”. However, Ca²⁺ binds to the enzyme-vanadate complex via sites that are only accessible from the interior of the SR vesicles. The complex Ca₂E*Van, which is rapidly formed, isomerizes very slowly (t_1/2 ~ 1 min) to the stable ternary complex. The mutual destabilization between bound vanadate and two bound Ca²⁺ ions is only 1.6 kcal/mol, much smaller than that produced by the interaction of calcium and phosphate.