CHARACTERIZATION OF A PROTON-TRANSLOCATING ATPASE IN A TONOPLAST-VESICLE FRACTION FROM CITRUS

Tonoplast vesicles were isolated from Valencia Late (Citrus sinensis (L.) cv. Osbeck) leaves by sucrose cushion density gradient centrifugation. Measurements of marker enzymes for microsomal membranes like latent IDPase (Golgi apparatus), Antimycin-A-insensitive NADPH-cytochrome c reductase (endoplasmic reticulum) and cytochrome-c-oxidase (mitochondria), and measurements of the ATP-hydrolysis activity with inhibitors specific for ATPases of different microsomes indicated a good purity of the tonoplast vesicle fraction. The rate of ATP-hydrolysis of the tonoplast H+-ATPase was optimal at pH 7.4. It was stimulated by Cl- with a maximum at 60 mM and was inhibited by 60 mM NO3-. The tonoplast H+-ATPase hydrolyzed [MgATP]2- in preference to other Mg-nucleotides in the sequence ATP > GTP > UTP greater-than-or-equal-to CTP > ADP. The apparent K(m) for [MgATP]2--hydrolysis was 0.38 +/- 0.04 mM. The H+-ATPase activity of tonoplast vesicles was sensitive to the anion channel blocker DIDS (I50 = 15 muM). Even in the absence of Cl- the rate of ATP hydrolysis was inhibited by 30 % at 40 muM DIDS, which indicates a direct effect of DIDS on the tonoplast H+-ATPase. Quinacrine-fluorescence quenching measurements of tonoplast vesicles showed that the tonoplast H+-ATPase was able to build up an MgATP-dependent electrochemical proton gradient across the vesicle membrane. This gradient broke down in the presence of the protonophore gramicidine A and by the inhibition of the tonoplast H+-ATPase by NO3-. Determination of latent H+-ATPase activity with 0.002 % Brij 58 showed that the tonoplast vesicle fraction contained approximately equal amounts of rightside-out vesicles and inside-out vesicles. In the tonoplast vesicle preparation five polypeptides (63, 52, 37, 30, and 16 kDa) cross-reacted with an antiserum against the tonoplast H+-ATPase from leaves of Kalanchoe daigremontiana. Our results indicate that the tonoplast vesicles of Citrus leaves contain an H+-ATPase with similar properties as the tonoplast H+-ATPase from other plant and fungal sources. This characterization of the enzyme is the basis for the investigation of the energetization and regulation of secondary active transport mechanisms of ions and metabolites at the tonoplast in response to environmental stress.