EXISTENCE OF HIGH-AFFINITY AND LOW-AFFINITY VANADATE-BINDING SITES ON CA2+-ATPASE OF THE SARCOPLASMIC-RETICULUM

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 0.1-mu-M, and the low-affinity sites one of 36-mu-M. 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 13-mu-M. 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.