The use of quench reagents for resolution of single transport cycles in sarcoplasmic reticulum.

ATP-dependent Ca2+ uptake, ATPase phosphorylation, and Pi production by sarcoplasmic reticulum vesicles were measured in rapid quench experiments, using trichloroacetic acid, H&+, EGTA, EDTA, and La3+ as quench reagents. Trichloroacetic acid, EDTA, and H&+ cause total inhibition of enzyme activity and loss of accumulated calcium. On the other hand, EGTA and La3+ permit retention of calcium accumulated by the vesicles, while blocking ATP utilization within a time shorter than a single enzyme cycle. While EGTA inhibits specifically enzyme phosphorylation by ATP, La3+ quenches both enzyme phosphorylation and phosphoenzyme cleavage. A significant time lag between block of phosphoenzyme formation and block of phosphoenzyme cleavage suggests that the latter effect requires penetration of La3+ inside the vesicles. ATPase activity is also blocked with EDTA, which can be used for comparative studies on quenching at neutral or acidic pH and detection of possible acidlabile intermediates. It is concluded that the information yield from rapid quench experiments can be greatly enhanced by selective use of reagents displaying specific quench mechanisms. Thereby, it is demonstrated directly that: (a) an initial burst of Ca2+ uptake following addition of ATP is due to a single turnover of enzyme units phosphorylated before quenching, and corresponds to a distinct translocation step occurring with K > 14 s-‘; (b) a step related to hydrolytic cleavage of the phosphorylated intermediate is rate-limiting in the enzyme turnover; (c) the concentration of acid-labile enzyme l phosphate complex is negligible in the presence of ATP and Ca’+; (d) modulation of enzyme activity is dependent on occupancy of cation binding sites exposed to the outer or inner side of the membrane, consistent with a model of shifting site orientation to account for vectorial transport: (e) a state of very low permeability and minimal efflux of intravesicular Ca2+ is obtained only in the presence of ATP and Mg’+, when Ca2+ is dissociated from high affinity ATPase sites.