Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.