Abstract: | The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PPiase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PPi-dependent H+ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H+ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H+ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PPi. This stimulation was observed only when the PPiase was active. A similar effect was not found for PPi-dependent H+ transport after pre-energization with ATP. Hence, a PPiase-activated ATP-dependent H+ transport can be distinguished from the basic ATP- and the basic PPi-dependent H+ transport. In parallel a PPi-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PPiase. PPiase-activated ATP-dependent H+ transport depends on the presence of permeant anions. It shows properties of both H+ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H+ transport activity, the nitrate stimulation and the KF inhibition of the PPi-dependent H+ transport activity. Only MgPPi and MgATP were effective as the respective substrates. The PPiase-activated ATP-dependent H+ transport had a half life of about 5–9 minutes. It is concluded that the PPiase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed. |