MATERIALS AND METHODSAn anion-sensitive ATP-dependent H transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of Recently, evidence has accumulated for the presence of two microsomal H+-ATPases in plants involved in the production of electrogenic H+ gradients (4,6,8,10, 11,18,19). One is associated with high density membranes (about 1.16-1.17 g/cm3 inhibited by vanadate and correlated with marker enzymes for plasma membrane (4,7,8,19). The second one is found in low density membranes (about 1.10 g/cm3), activated by Cl-, inhibited by NO3 and is thought to be of tonoplast origin (4,9,16,17,19). This latter assumption is strongly supported by recent results obtained with tonoplast vesicles prepared from previously isolated vacuoles (4) which show the same characteristics. Solubilization (20) and 'negative purification' (7) of both ATPases have been attempted which will help to further elucidate their reaction mechanism. Meanwhile, comparatively little efforts were undertaken to characterize the substrate kinetics (1, 9) of the tonoplast-type ATP-dependent H+ transport although a kinetic regulation ofthe H+ transport ATPases seemed likely (4).In this study, we partially characterize a tonoplast-type ATPdependent H+ transport in microsomal membranes (4,25). The disappearence of unprotonated neutral red in the reaction medium which reflects the accumulation of the protonated species in the membrane vesicles (14) is used to monitor H+ transport. As free protonated neutral red is in equilibrium with membranebound protonated neutral red with different spectral characteristics (13), it is not opportune to follow the accumulation of the protonated species. We present evidence for a competitive inhibition ofATP-dependent H+ transport by ADP By that time, the coleoptiles had an average length of 4 cm. The coleoptiles were cut off in room light with a razor blade 2 mm from the node, and the primary leaves were removed. After collection on ice cold deionized H20, they were filtered and blotted dry on filter paper. After determination of fresh weight, they were immediately immersed into the medium for plasmolysis (20 mM Mops' 400 mm sucrose, adjusted to pH 7.2 with 0