Two alternate kinetic routes for the decomposition of the phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase.

The decomposition of the phosphorylated intermediate (EP) of sarcoplasmic reticulum ATPase, purified by the method of deoxycholic acid extraction, was studied by first phosphorylating with [gamma-32P]ATP, then diluting the reaction mixture with 20 volumes of medium containing nonradioactive ATP, and finally quenching serial samples with acid for determination of residual [32P]EP. The time course of [32P]EP decomposition consists of an initial fast phase followed by a slow phase. The two components of EP, EPfast (1.1 nmol/mg) and EPslow (2.8 nmol/mg), decomposed with the rate constants of 6 and 0.8 min-1, respectively, in the presence of 0.5 mM CaCl2, 5mM MgCl2, and 90 mM KCl at pH 7.0 and O degrees C. The sum of the hydrolytic activities corresponding to the two components accounts for the steady state velocity of the Pi production under the same conditions, indicating that the two components represent simultaneous pathways, rather than sequential steps of EP decomposition. As the time of phosphorylation with [gamma-32P]ATP is increased from 2 to 15 s, the fraction of EPfast decreases in favor of EPslow. This conversion decreases the rate of total Pi production by the enzyme following an initial Pi burst. Conversion of EPfast to EPslow is favored by millimolar concentrations of Ca2+. On the other hand, conversion of EPslow to EPfast is obtained by reducing Ca2+ or raising Mg2+ concentration, but is prevented by removal of ADP. The EPslow fraction decreases in favor of EPfast as the temperature is increased from 0 to 22 degrees C.