Role of the ATPase of sugar-cane vacuoles in energization of the tonoplast.
Vacuoles of sugar-cane suspension cells contained a tonoplast-bound ATPase which was exclusively located on the cytoplasmic side of the vacuole. Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase. ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast-bound GDP-hydrolyzing and GTP-hydrolyzing enzymes which can interfere with the ATPase assay. Other phosphate anhydrides and esters were not hydrolyzed. The addition of MgATP polarized the tonoplast from about 0 mV to an interior-positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential. The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m-chlorophenylhydrazone. These data are interpreted as evidence for the action of tonoplast-bound ATPase as a pump which translocates protons into the vacuoles. The activity of the ATPase was highly specific for MgATP2-; the other important ionic states of ATP:ATP4-, HATP3-, MgHATP-, and Mg2ATP neither stimulated nor inhibited. The same was true for Mg2+. Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast-ATPase may pick up the proton for translocation from the cytoplasm. The saturation kinetics for MgATP2- hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D050260:Carbohydrate Metabolism; D004735:Energy Transfer / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D010944:Plants / Q000201:enzymology*; D011522:Protons; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology
D011522:Protons; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium