Transient Kinetics of Formation of Phosphorylated Intermediate(EP) by Fragmented Sarcoplasmic Reticulum From Bullfrog Skeletal Muscle: Ligand Addition Sequence-Dependent Rate of EP Formation

Abstract

In order to obtain a better understanding of Ca²⁺-activated ATP hydrolysis by sarcoplasmic reticulum, the transient kinetics of phosphorylated intermediate (EP) formation was examined with different sequences of addition of Ca²⁺ and ATP to GEDTA-added (Ca²⁺-free) fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. With a short delay line (10-20ms), the addition sequence of ATP followed by Ca²⁺ gives rise to a faster EP formation without any lag time. In the reverse sequence of ligand addition, a lag time of 2.5-3ms was consistently observed irrespective of ATP concentration, and the rate of EP formation was lower. As the preincubation time with Ca2+ became longer, the rate constant for EP formation and the maximum level of EP attainable increased even in the presence of fixed concen-trations of Ca²⁺ and ATP. The rate constant per the unit concentration of EP, however, remained constant, indicating second-order kinetics between Ca²⁺-activated FSR and ATP. With a preincubation time of 83.6ms, no lag time was observed. In the addition sequence of ATP and 83.6ms later Ca²⁺, a burst in EP formation was observed, which was followed by a usual exponential time-course of EP forma-tion. Similar determinations in the presence of various concentrations of ATP led us to the following estimates of the rates of the reactions leading to EP formation at 15°C.
The ATP-activation route is much faster and more relevant to the physiologically important Ca-uptake process.