Equilibrium and kinetic fluorescence methods have been used to characterize the interactions between K+ and the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. K+ shifts the E2-E1 equilibrium of the ATPase towards E1 and increases the rate of Ca2+ binding to the ATPase, as detected by changes in tryptophan fluorescence intensity, suggesting that K+ increases the rate of the E2-E1 transition. The data are consistent with binding of K+ at the inner Ca(2+)-binding site on the ATPase in competition with H+ and Mg2+, with a higher affinity in the E1 than in the E2 conformation. K+ has no effect on the affinity for Mg2+, as detected by changes in tryptophan fluorescence intensity; since it has been proposed that the changes in tryptophan fluorescence follow from binding to Mg2+ at the outer Ca(2+)-binding site, this suggests that K+ is unable to bind at the outer Ca(2+)-binding site. K+ increases the rate of dissociation of Ca2+ from the Ca(2+)-bound ATPase and reduces the effect of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It is suggested that these effects of K+ are the result of binding at a 'gating' site on the ATPase, in competition with binding of H+. Binding of K+ at the inner Ca(2+)-binding site and at the gating site account for the observed effects of K+ on the affinity of the ATPase for Ca2+.

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.

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.

On reconstitution of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC] or dinervonylphosphatidylcholine [di(C24:1)PC] the stoichiometry of Ca2+ binding changes from the usual two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound per ATPase molecule. For the ATPase in di(C24:1)PC, removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity, as observed for the ATPase in dioleoylphosphatidylcholine [di(C18:1)PC]. For the ATPase in di(C14:1)PC removal of Ca2+ results in no change in tryptophan fluorescence intensity. In the presence of Mg2+, removal of Ca2+ from the ATPase in di(C18:1)PC or di(C24:1)PC results in a decrease in tryptophan fluorescence intensity, but for the ATPase in di(C14:1)PC this results in an increase in intensity. Fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) is the same for the ATPase in di(C18:1)PC or di(C24:1)PC, but is markedly greater in di(C14:1)PC, consistent with a 4-fold increase in the E1/E2 equilibrium constant. Addition of Mg2+ to NBD-labelled ATPase in di(C18:1) PC or di(C24:1)PC results in an increase in NBD fluorescence, attributed to stronger binding of Mg2+ to the E1 than to the E2 conformation; addition of Mg2+ had no effect on the fluorescence of the NBD-labelled ATPase in di(C14:1)PC. In the absence of Ca2+, Mg2+ increased the tryptophan fluorescence of the ATPase in di(C14:1)PC, di(C18:3)PC or di(C24:1)PC, with the same binding-constant for Mg2+ in all three lipids. Addition of Mg2+ to the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin resulted in a decrease in fluorescence in di(C18:1)PC or di(C24:1)PC but had no effect in di(C14:1)PC. These effects are interpreted in terms of binding of Ca2+ at a single outer Ca2+ binding-site on the ATPase in di(C14:1)PC and di(C24:1)PC, in a conformation in which the inner site is occluded [in di(C14:1)PC] or modified in its affinity for Ca2+ [in di(C24:1)PC]. Thapsigargin binds to the ATPase, reducing its affinity for Ca2+ both in di(C14:1)PC and di(C24:1)PC.

The fluorescence intensity of the Ca# + -ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with P i , this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of P i fix the value of the equilibrium constant for the phosphorylation reaction E2PMg 8 E2P i Mg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show