High-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca 2+-ATPase labeled with fluorescein isothiocyanate

Abstract

Conditions were found that allowed both the fluorescence detection of vanadate binding to the Ca 2+-ATPase of skeletal muscle sarcoplasmic reticulum and the vanadate-induced formation of two-dimensional arrays of the enzyme. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca 2+-ATPase increased with high-affinity vanadate binding ( K a = 10 6 M −1 ) as reported by Pick and Karlish (Pick, U. and Karlish, S.D. (1982) J. Biol. Chem. 257, 6120–6126). The Ca 2+ and Mg 2+ dependencies for high-affinity vanadate binding were similar but not identical to those for orthophosphate. In addition, it was found that there is low-affinity ( K a = 380 M −1 ) vanadate binding, which causes a 25% decrease in fluorescence. The Ca 2+ and Mg 2+ dependencies of the low-affinity vanadate binding were different from those of orthophosphate or high-affinity vanadate binding. The covalent attachment of fluorescein isothiocyanate (FITC) in the ATP site of the Ca 2+-ATPase did not affect the formation of two-dimensional arrays, as detected by negatively stained electron micrographs. Vanadate concentrations high enough to saturate the low-affinity binding caused two-dimensional arrays as reported by Dux and Martonosi (Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599–2603). In addition, freeze-fracture replicas of quick-frozen specimens showed rows of indentations in the inner leaflet of the bilayer that corresponds to the arrays seen on the outer leaflet. This appearance of indentations suggests that low-affinity vanadate binding causes a transmembrane movement of the Ca 2+-ATPase. By contrast, high-affinity vanadate binding was shown to cause neither array formation nor the appearance of indentations.