Existence of High- and Low-Affinity Vanadate-Binding Sites on Ca²⁺-ATPase of the Sarcoplasmic Reticulum

Abstract

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca²⁺-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1μM, and the low-affinity sites one of 36μM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca²⁺-ATPase. These results indicate that 1 mol of Ca²⁺-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxyethylene-9-laurylether (C₁₂E₉), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13μM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca²⁺-ATPase. Vanadate binding to the solubilized Ca²⁺-ATPase was competitively inhibited by inorganic phosphate. When the detergent was removed to reconstitute SR membrane, vanadate binding again exhibited a biphasic concentration dependency curve. These results indicate that interactions between the ATPase molecules in intact SR membranes may involve the cooperative binding of vanadate to the enzyme.