Kinetics of adenosine triphosphate synthesis in bovine heart submitochondrial particles

The kinetics of ATP synthesis by submitochondrial particles were studied with a continuous flow mixing technique allowing measurements of ATP synthesis between 15 and 120 ms after mixing for estimation of the initial rate. During the initial 100 ms, ATP synthesis proceeded at a faster rate when driven by an artificially imposed electrochemical proton gradient than when driven by the respiratory substrate NADH. The rate of ATP synthesis driven by the artificial electrochemical proton gradient was dependent upon the magnitude of the gradient, and was affected similarly by variations of either the pH gradient or membrane potential. Initiation of ATP synthesis driven by NADH oxidation with ADP plus Pi resulted in an initial rate of phosphorylation equivalent to that during steady state respiration. However, a lag in phosphorylation was observed when ATP synthesis was initiated by oxygen or NADH. Valinomycin caused transient uncoupling of ATP synthesis driven by respiration since it strongly inhibited ATP synthesis during the initial 100 ms but it had no effect during steady state phosphorylation. The kinetic results strongly suggest that an electrochemical proton gradient is an obligate intermediate between electron transport and ATP synthesis on the main pathway of energy transduction.