Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells (original) (raw)
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Characterization of a Proton-Translocating ATPase in Microsomal Vesicles from Corn Roots
Plant Physiology, 1982
Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg2-ATPase activity of the sealed vesicles was stimulated by Cl-and NHIt and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The lonophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 65. The ATPase was inhibited by N03-, was insensitive to K+, and was not inhibited by 100 micromolar vanadate or by 1 milfimlar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by ClI, inhibited by N03-, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATPdependent fluorescence quench both required a divalent cation (MgNW 2 Mn2" > Co2") and were inhibited by high concentrations of Ca2". The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the protontranslocating ATPase differed from those of the mitochondrial F1F,-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. ATP-dependent proton transport and ATP-dependent generation of membrane potentials have been demonstrated in microsomal vesicles obtained from several plant tissues. Methylamine uptake (16), imidazole uptake (22), shift in wavelength of neutral red dye (9, 10), and quenching of fluorescence of quinacrine (3, 6, 7), 9-amino acridine (3), and acridine orange (26) have all been used to demonstrate ATP-dependent acidification of the interior of microsomal vesicles from higher plants. SCN-uptake (19, 22, 25) and shift in absorbance spectrum of the membrane-potentialsensitive dye oxanol (3) have been used to demonstrate generation of a positive membrane potential, discharged by protonophores, in the microsomal vesicles. The ATPase activity of the vesicles is stimulated by ionophores (3, 6, 7, 20, 21, 23-25). Thus, there is now excellent evidence for the presence of an electrogenic, protontranslocating ATPase which utilizes externally supplied ATP to 'Supported by Grant PCM 78-12119 from the National Science Foundation to R. M. Spanswick.
PLANT PHYSIOLOGY, 1987
ABSTRACr Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca2 transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca2". A low affinity Ca2" uptake system (K., > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H'/Ca2" antiport. A high affinity Ca2" uptake system (K. = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca2" transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca2f/H antiport activity could only by partially ascribed to an effect of low temperature on H-ATPase activity, ATP-dependent H' transport, passive H' fluxes, or passive Ca2" fluxes.
Characterization of a Proton-Translocating ATPase in a Tonoplast-Vesicle Fraction from Citrus
Journal of Plant Physiology, 1993
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 60mM and was inhibited by 60mM N0 3-. The tonoplast H+-ATPase hydrolyzed [MgATP]2-in preference to other Mg-nucleotides in the sequence ATP> GTP > UTP ~ CTP > ADP. The apparent Km for [MgATP]2-hydrolysis was 0.38 ± 0.04mM. The H+-ATPase activity of tonoplast vesicles was sensitive to the anion channel blocker DIDS (Iso = 15 J.lM). Even in the absence of Cl-the rate of ATP hydrolysis was inhibited by 30 % at 40 J.lM 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 N0 3-. 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 16kDa) 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.
Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells
Acta Physiologiae Plantarum, 2001
Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifngation of microsome pellet (10,000-g0,000 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 PPi, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H+PPiase.
Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles
PLANT PHYSIOLOGY, 1985
Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATPdriven H"-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H-transport. At saturating (3 millimolar) concentrations of Mg2":ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H'transport, but had no effect on ATP-driven transport. Moreover, PPidependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO3, unlike the ATPdependent Hf-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound HW-translocating pyrophosphatases. Both potassium and a permanent anion (NO3-> Cl-), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N'-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.
Ontogenez
Comparative analysis of the transport activity of proton pumps (plasmalemma H+-ATPase, vacuolar H+-ATPase, and vacuolar H+-pyrophosphatase) in the membrane preparations obtained from coleoptile cells ofetiolated maize seedlings (Zea mays L.) was carried out. The highest level ofvacuolar pyrophosphatase activity was observed during the early development of coleoptile cells under growth intensification through the elongation. The role of ATPase pumps of tonoplast and plasmalemma in the transport of hydrogen ions increases during further development. The plasmalemma activity in this process is higher. When the growth stops, the activity of proton pumps becomes significantly lower. Nevertheless, their substrate specificity and sensitivity to proton pump inhibitors do not change, which can be an evidence of physiological significance of pumps in the maintenance of cell homeostasis.
Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes
PLANT PHYSIOLOGY, 1987
The effects of NO3and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H+-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KN03 when assayed at 24°C or above and was tentatively identified as the tonoplast H+-ATPase. A smaller peak of H+-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KN03 and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H'-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO3. The time course for fluorescence quench indicated that addition of ATP in the presence of KN03 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KN03 was explained by the temperature-dependent delay of the inhibition by KNO3. The plasma membrane H'-ATPase maintained a pH gradient in the presence of KN03 for up to 30 minutes at 24°C. ' Mention of a specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products. 526
Effects of pH on proton transport by vacuolar pumps from maize roots
Physiologia Plantarum, 1992
Protons pumps of the tonoplast may be involved in the regulation of cytosolic pH, but the effects of pH on the coupled activities of these transporters are poorly understood. The effects of pH on the activities of the H^-translocating pyrophosphatase (PP^ase) and vacuolar-type H*-translocating adenosine tdphosphatase (H*-ATPase) from maize {Zea mays L. cv. FRB 73) root membranes were assessed by changes in acridine orange absorbance, and the data were ana!yzed according to a mode! that simu!taneous!y considers proton transport by the pump and those processes that reduce net transport. The addition of either pyrophosphate or ATP to either mierosomal or tonoplast membranes generated a pH gradient. The pH gradient generated in the presence of both substrates was not the sum of the gradients produced by the two substrates added separate!y. When membranes were separated by sucrose density gradient centrifugation, pyrophosphate (PPJ-dependent proton transport was associated with light density membranes having tonopiast H*-ATPase activity. These results indicate that some portion of the PPjase was located on the same membrane system as the tonop!ast ATPase; however. tonop!ast vesicles may be heterogeneous, differing slightly in the ratio of ATP-to PPj-dependent transport. Proton transport by both the PP,ase and ATPase had maximal activity at pH 7.0 to 8.0. Decreases in proton transport by the ATPase at pH above the optimum were associated with increases in the processes that reduce net transport. Such an association was not observed at pH values below the optimum. These results are discussed in terms of in situ regulation of cytoplasmic pH by the two pumps.
The tonoplast proton—Translocating ATPase of higher plants as a third class of proton—Pumps
Biochimie, 1986
-Taken together, all the data reported recently in the literature suggest that tonoplast ATPase belongs to a new class of proton pumps. To date, the most studied system is the proton-pumping ATPase from the tonoplast of Hevea latex. Its main characteristics are presented. It resembles the mitochondrial ATPase in its specificity, its substrate affinity, and its sensitivity to different inhibitors. However, for some aspects, it resembles the plasma membrane system in its response to other inhibitors tested (quercetin for example). It differs from both ATPases in its sensitivity to nitrate as well as by its molecular structure, i.e. a complex exhibiting a least 4 or 5 polypeptides. These results favor the existence of a third class of proton pumps, intermediate between the FIF0-class and the EiE2-class. tonoplast I lutoidal membrane / proton pump / subunits R6sum6-L'ATPase tonoplaslique des plantes sup~rieures apparfient h une troisi~me classe de pompe-~t-proton. Le systbme le mieux ddcrit actuellement est i'ATPase tonoplastique du latex d'Hevea. Son dtude ne se heurte pas aux difficultds rencontrdes chez les autres vdgdtaux: leur faible quantitd, leur contamination par d'autres structures membranaires et une ddnaturation non contr61de d l'origine de nombreux rdsultats difficiles d interprdter. Les propridtds de cette activitd pompe-d-proton sont dvoqudes. Elle s'avbre trbs proche de l'activitd mitochondriale en considdrant une partie de ses propridtds: son affinitd pour le substrat, sa spdcificitd et sa sensibilitd d certains inhibiteurs caractdristiques des A TPases du type FoF l (trimethyltin, DCCD). De plus, elle est sensible aux protonophores. Mais, par d'autres aspects, elle se rapproche des A TPases du type EIE 2. Dbs lors, tout contribue d penser que ce type d'A TPase appartiendrait D une troisikme classe de pompesd-protons, intermddiaire entre les deux classes actuellement ddcrites. Les rares dtudes faites sur la structure moldculaire de cette activitd solubilisde et purifide le confirment. Par ailleurs, de par leurs propridtds cornparables d celles ddcrites pour l'ATPase tonoplastique, toutes les ATPases situdes sur les membranes des compartiments endocellulaires appartiendraient d cette nouvelle classe de pompe-d-protons. tonoplaste I membrane lutoidique I pompe-?l-protons I sous-unit~s This review represents part of the seminar given on this topic during the