title: Existence of high- and low-affinity vanadate-binding sites on Ca(2+)-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 Ca(2+)-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 microM, and the low-affinity sites one of 36 microM. 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 Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-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 polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. 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 Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biology, Faculty of Science, Osaka University.
authors.forename: K
authors.initials: K
authors.lastname: Yamasaki
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012995:Solubility; D014638:Vanadates / Q000378:metabolism*
chemical_list: D010710:Phosphates; D014638:Vanadates; D000252:Calcium-Transporting ATPases