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 Phase

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, k_d, 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 k_d 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 k_d 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, k_d, 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 k_d was given by
k_d=(k_{d, initial}-k_{d, steady})×exp(-k_tr•t)+k_{d, steady},
k_{d, initial}/k_{d, steady}=7.3, and k_tr=0.5sect^-1, where k_{d, initial} and k_{d, steady} are the rate constant of E_??_P decomposition immediately after starting the reaction and that in the steady state, respectively. The value of k_tr is the rate constant for transition in the k_d 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 k_d 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 k_d 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.