TIME-RESOLVED MEASUREMENTS SHOW THAT PHOSPHATE RELEASE IS THE RATE-LIMITING STEP ON MYOFIBRILLAR ATPASES

The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, ther e is a burst of P-i formation, due to AM . ADP . P-i or P-i, as shown in the scheme: AM + ATP <-> A . M . ATP <-> AM . ADP . Pi <-> AM . ADP + P-i <-> AM + ADP. Therefore, in the steady state, either AM . ADP . P-i or AM ADP or both predominate. To determine which, we studied the reaction using a P-i binding protein (from E. coli) labeled with a fl uorophore such that it is specific and sensitive to free P-i [Brune, M . et al. (1994) Biochemistry g 33, 8262-8271]. We show that the P-i bu rsts with myofibrillar ATPases (calcium-activated or not, or crosslink ed) are due entirely to protein bound P-i. Thus, with myofibrillar ATP ases the AM . ADP . P-i state predominates.