A Time lag in the onset of ATP- Pi exchange catalyzed by purified ATP-synthase (CF 0-CF 1) proteoliposomes and by chloroplasts

Abstract

The time course of ATP- P i exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made, ( i) The onset of 32 P i incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 °C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate, ( ii) The initial lag is independent of Mg-ATP concentration in the range 0.2–5 m m and of the presence of ADP. In contrast, the steady-state rate of ATP- P i exchange has an apparent K m of 0.3 m m for Mg-ATP and is stimulated by ADP. ( iii) Increasing the temperature from 30 to 45 °C decreases the lag from 6 min to zero. The steady-state rate of ATP- P i exchange is affected to a much smaller extent by the temperature in this range, ( iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate, ( v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system, ( vi) Light-triggered ATP- P i exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP- P i exchange does not reflect the time required for the buildup of a protomotive force (Δ − μ H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an “exclusive ATPase state” to a “reversible ATP-synthase state”. This slow transition is not directly coupled to a transmembrane pH or potential gradient.