Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H⁺-ATPase Antibodies

Abstract

ABSTRACIA tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifuption of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrateinhibited proton pumping activity coincided with a Mg2"-ATP dependent accumulation of 3-0-methyl-D-glucose (OMG) as determined by a membrane filtration technique using '4C-labeled substrate. OMG transport showed an apparently saturable component with a K,. of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide mchlorophenylhydrazone. Polyclonal antibodies aganst solubilized native tonoplast H+-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62. kilodalton subunit had only a slight effect on both processes.

The Use of Antisense mRNA to Inhibit the Tonoplast H + ATPase in Carrot

“…ATP-dependent 14 C-OMG uptake by wild-type and (3-glucuronidase (GUS) control cells was complete by 40 min, after which a decrease was observed, probably due to vesicle leakage. The rates of ATP-driven 14 C-OMG uptake are similar to those observed previously in other systems (Rausch et al, 1987) tonoplast vesicles isolated from the two antisense cell lines was negligible, in agreement with the inhibition of proton pumping activity shown in Figure 3. The slightly higher levels of 14 C-OMG uptake by the ncr cell line is consistent with the slightly higher levels of H + -pumping activity measured in these membranes.…”

Influence of Plant Growth at High CO2 Concentrations on Leaf Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Intracellular Distribution of Soluble Carbohydrates in Tobacco, Snapdragon, and Parsley

“…at their resolution limits). This result is not unreasonable, since leaf vacuolar hexose transporters (Rausch et al, 1987) and hexokinases (Schnarrenberger, 1990) typically have K , values of about 100 p~. That we did not observe any enhanced cytosolic hexoses in tobacco grown at high CO, still is not inconsistent with the proposed function of cytosolic hexokinase as a sugar sensor (Jang and Sheen, 1994;Jang et al, 1997), since cytosolic hexose metabolism may be affected by futile cycling of Suc (Geigenberger and Stitt, 1991) or since levels of cytosolic hexoses possibly could be higher at other times during the day (e.g.…”

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or apoplasm

“…Guy et al (1979) reported an uptake of D-glucose and 3-0-methyl-D-glucose into isolated vacuoles of pea leaves, which was slightly stimulated by ATP and inhibited by protonophores. Rausch et al (1987) showed an ATPdependent uptake of 3-O-methyl-D-glucose into vesicles from tonoplast membranes of Zea mays coleoptiles, which had a Km-value of 110 pM and resulted in an accumulation by a factor of two. On the other hand, Martinoia et al (1987) could not find any stimulating effect of ATP on the glucose uptake in vacuoles of barley protoplasts.…”