Binding of Ca2+ to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies

Abstract

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1'Ca2. 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, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1'Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1'Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the 'gating' site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.

Evidence for direct involvement of the sarcoplasmic reticulum Ca2+‐ATPase in a passive monovalent cation (K+/Na+) exchange

“…On the other hand, inhibition by vanadate and TG would result in locking the enzyme in a stable 'E2' conformation. Two characteristics of the EI-E 2 equilibrium are in favor of this scheme: the rate of Ez-E 1 is accelerated at basic pH [23][24][25] and by monovalent cations [6,26,27]. In addition the evaluation of the K ÷ or Na + flux rate gives a value of turn-over in the same range as the rate of the E2--+E1 conformational change.…”

Functional Consequences of Alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the Interfaces between Domain P, M3, and L6-7 of Sarcoplasmic Reticulum Ca2+-ATPase

“…Hence, it can be shown that the K 0.5 for vanadate inhibition equals K V ϫ (1 ϩ 1/K 12 ), where K V is the dissociation constant describing vanadate interaction with E2, and K 12 is the E1-E2 equilibrium constant [E2]/[E1]. Because K 12 is ϳ1 for wild type under the conditions applied here at neutral pH in the absence of Ca 2ϩ (29,40), the K 0.5 for vanadate inhibition of wild type must be close to 2 ϫ K V , and the expected K 0.5 for vanadate inhibition of a mutant in which K 12 is increased 58-fold equals K V ϫ (1 ϩ 1/58) Ϸ K V , thus corresponding to a 2-fold decrease, relative to wild type.…”

Ca2+ Release from the Phosphorylated and the Unphosphorylated Sarcoplasmic Reticulum Ca2+ ATPase Results in Parallel Structural Changes

“…However, several partial reactions involving the cytoplasmic high affinity sites are unaffected by 20 -40 mM lumenal Ca 2ϩ (26,30,31), indicating that such a transition may not occur.…”