Competition between decavanadate and fluorescein isothiocyanate on the Ca2+-ATPase of sarcoplasmic reticulum.

Abstract

The binding of vanadate and fluorescein isothiocyanate to the Ca2+-transport ATPase of sarcoplasmic reticulum (EC 3.6.1.3) was analyzed. Monovanadate binds to the Ca2+-transport ATPase at a single high affinity site (site 1), that is presumably related to the binding site for inorganic orthophosphate, and to one of the two sites for decavanadate. Binding of vanadate to this site stabilizes the enzyme in the E2 conformation, with inhibition of ATPase activity and the formation of crystalline arrays of Ca2+-ATPase. Decavanadate also binds with high affinity to a second site on the Ca2+-ATPase (site 2), that is blocked by fluorescein isothiocyanate and may be part of the binding site for ATP. Crystallization of Ca2+-ATPase in sarcoplasmic reticulum, labeled with fluorescein isothiocyanate, by either monovanadate or decavanadate implies that occupation of site 1, but not of site 2, by vanadate is required for the conformational change of Ca2+-ATPase leading to the formation of crystalline arrays.