BINDING OF CA2-MG2+)-ATPASE OF SARCOPLASMIC-RETICULUM - EQUILIBRIUM STUDIES( TO THE (CA2+)

Equilibrium fluorescence methods have been used to establish a model f or Ca2+ binding to the (Ca2+-Mg2+)-ATPase of skeletale the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 reversib le arrow E1 <reversiblearrow>E1Ca reversible arrow E1'Ca reversible ar row E1'Ca-2. The E1 conformation of the ATPase initially has one high- affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca2+-binding site on the ATPase is formed after binding of Ca2 to the first, inner, site on E1 and the E1Ca reversible arrow E1'Ca c onformation change. The pH- and Mg2+-dependence of the E2 reversible a rrow E1 equilibrium has been established after changes in the fluoresc ence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reti culum can bind to the first Ca2+-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after b inding of Ca2+ follows from the E1Ca reversible arrow E1'Ca change. Th e pH- and Mg2+-dependence of this change defines H+- and Mg2+-binding constants at the two Ca2+-binding sites. It is proposed that the chang e in tryptophan fluorescence observed on binding Mg2+ follows from bin ding at the second Ca2+-binding site. Effects of pH and Mg2+ On the fl uorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxyco umarin are proposed to follow from binding to a site on the ATPase, th e 'gating' site, which affects the affinity of the first Ca2+-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the AT Pase.