Determination of coupling ratios of the calcium pump of sarcoplasmic reticulum by pulse methods

Coupling of Ca2+ transport to ATP hydrolysis in isolated sarcoplasmic reticulum vesicles has been studied following pulsed additions of either ATP or Ca2+. ATP was infused as a pulse into medium, whose free Ca2+ concentration was maintained constant at saturating levels by a calciumstat procedure, using either a Ca2+-selective electrode or the spectrophotometric arsenazo III technique as Ca2+ indicators. The low ATP levels virtually exclude contributions by "basal" ATPase activity. Passive leakage of Ca2+, monitored after an ATP pulse, does not contribute more than 5% to subintegral coupling ratios. Pulsed additions of Ca2+ were made into medium. containing saturating concentrations of ATP, whose hydrolysis was monitored by a pH-stat procedure. Ca2+-stimulated hydrolysis continued until all the Ca2+ was transported into the vesicles. Values for the coupling ratio, Ca2+/ATP, of 1.82 +/- 0.12 and 1.79 +/- 0.15 were obtained by the ATP- and Ca2+-pulse methods, respectively.