Reaction Mechanism of the Ca2+-dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle: XI. Re-evaluation of the Transition of ATPase Activity during the Initial Phase1

Publisher Website
Google Scholar
Abstract
We previously reported (J. Biochem. 70, 95–123 (1971)) that the time course of P1 liberation in the reaction of Ca2+, Mg2+-dependent ATPase [EC 3. 6.1. 3] of fragmented sarcoplasmic reticulum (SR) consists of a lag phase, a burst phase, and a steady phase. We also showed that the rate constant, kd, of decomposition of the phosphorylated intermediate (E˜P) decreases during the initial phase, and suggested that the burst phase is due to transition of the kd value. Recently, Froehlich and Taylor (J. Biol. Chem. 250, 2013–2021 (1975)) claimed that the P1 burst is caused by the formation of an acid-labile intermediate containing phosphate (E.P) formed by rapid hydrolysis of E˜P. In the present study, the transition of the kd value during the initial phase was measured precisely, and the results showed that the burst phase is due to a transition in the kd value, not to the existence of E. P. The main results obtained were as follows: 1. After the SR had been phosphorylated with [γ-32P]ATP in the presence of Mg2+ and Ca2+ ions, further phosphorylation was stopped by the addition of EGTA. The concentration of E˜32P then decreased exponentially with time. 2. The first-order rate constant, kd, of decomposition of E˜32P after adding EGTA decreased with increase in the interval, t, between the start of E˜32P formation and the time of adding EGTA. The value of kd was given by kd = (kd, initial—kd, steady) × exp (−ktr.t) + kd, steady,kd, initial/kd, steady = 7.3, and ktr = 0.5 sec−1, where kd, initial and kd, steady are the rate constant of E˜P decomposition immediately after starting the reaction and that in the steady state, respectively. The value of ktr is the rate constant for transition in the kd value. 3. The value of kd was also obtained from the time course of E˜32P decomposition after adding a large amount of unlabelled ATP to stop E˜32P formation. The kd value thus obtained was the same as the value obtained by stopping E˜P formation with EGTA. 4. The observed time course of P1 liberation after adding ATP to the SR was interpreted quantitatively in terms of the observed time course of E˜P formation and the value of kd given above. Furthermore, we previously showed that when ADP was added to E˜P, ATP was formed in an amount equal to the amount of decrease in E˜P. Therefore, the possibility that in the forward reaction of SR-ATPase an appreciable amount of E. P exists in equilibrium with E˜P is excluded.

This publication has 0 references indexed in Scilit: