The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation.
Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism* / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D014639:Vanadium; D014638:Vanadates; D000252:Calcium-Transporting ATPases; D002118:Calcium