Abstract
In comparative experiments with Ca2+ ATPase in native sarcoplasmic reticulum vesicles and reconstituted proteoliposomes, we find that a variable stoichiometry of Ca2+ or Sr2+ transport per ATPase cycle is observed in the absence of passive leak through independent channels. The observed ratio is commonly lower than the optimal value of 2 and depends on the composition of the reaction mixture. In all cases, a progressive rise in the lumenal concentration of Ca2+ and Sr2+ is accompanied by a parallel reduction of coupling ratios. Significant ATPase activity remains even after asymptotic levels of Ca2+ accumulation are reached. This residual activity subsides if the Ca2+ concentration in the outer medium is reduced below activating levels (as it would following Ca2+ transients in muscle fibers). The reduction of stoichiometric coupling is explained with a reaction scheme, including a branched pathway for hydrolytic cleavage of phosphorylated intermediate before release of Ca2+ into the lumen of the vesicles. Flux through this pathway is favored when net lumenal Ca2+ dissociation from the phosphoenzyme is impeded and results in P(i) production accompanied by lumenal and medium Ca2+ exchange. Occurrence of reactions through branched pathways may have general implications for the stoichiometric efficiency of energy-transducing enzymes.
Highlights
Ratios of2 Ca2 +/ATP utilized have been observed under conditions permitting free Ca2 + to remain low in the lumen of the vesicles: (a) in steady state experiments in which oxalate is used for complexation oflumenal Ca2 + (Martonosi and Feretos, 1964) and (b) in pre-steady state experiments in which lumenal Ca2 + has yet to rise (Inesi et al, 1978)
Upon addition of Ca2 +, the rate ofATP hydrolysis rises in parallel with Ca2+ accumulation by the vesicles (Hasselbach, 1964). In this type of experiment, the oxalate added to the reaction mixture precipitates Ca2 + as it rises in the lumen of the vesicles and limits the free lumenal Ca2 + concentration to the calcium oxalate dissociation constant «0.1 mM)
We find that the stoichiometric ratio of Ca2+ uptake and RATIO OF CONSTANTS: 1.52+E07
Summary
Ratios of (or nearly 2) Ca2 +/ATP utilized have been observed under conditions permitting free Ca2 + to remain low in the lumen of the vesicles: (a) in steady state experiments in which oxalate is used for complexation oflumenal Ca2 + (Martonosi and Feretos, 1964) and (b) in pre-steady state experiments in which lumenal Ca2 + has yet to rise (Inesi et al, 1978). Ca2 +/ATP ratios lower than 2 are commonly observed under other conditions permitting lumenal Ca2 + to rise. Most diagrams represent the ATPase cycle with a pathway of sequential reactions which, in principle, do not allow uncoupling ofCa2 + transport and ATP utilization (Fig. 1, solid line). The uncoupling is accounted for by a branched pathway (Fig. 1, dotted line) of the ATPase cycle, permitting cleavage of the phosphorylated intermediate before net release of Ca2 + into the lumen of the vesicles
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