Interaction between poly(L-lysine) and membranes inhibits proton pumping by corn root tonoplast H+-ATPase

The influence of poly(L-lysine) binding on the coupled activities of nitrate-sensitive H⁺-ATPase in isolated corn ( Zea mays L. cv. FRB73) root tonoplast vesicles was investigated. The addition of membrane-impermeable poly(L-lysine) caused a slow increase in light scattering of the tonoplast suspension. Electron microscopy showed that the increase was the result of an aggregation of the vesicles. In the presence of 75 m M KCl, a concentration sufficient to sustain near optimal ATP hydrolysis, poly(L-lysine) slightly enhanced the hydrolysis activity but significantly inhibited proton pumping of the H⁺-ATPase. Inhibition increased with the average molecular mass of poly(L-lysine) and reached a maximum at 58 kDa. When total osmolarity was kept constant, the replacement of sucrose by KCl enhanced both ATP hydrolysis and proton pumping activities. However, enhancement of proton pumping was significantly greater than that of ATP hydrolysis. An increase in KCl, but not K₂SO₄, significantly relieved poly(L-lysine)-induced inhibition of proton pumping. Kinetic analysis indicated that poly(L-lysine) did not significantly affect the proton leakage of the tonoplast membranes under different energetic conditions. These results suggest that the electrostatic interaction between poly(L-lysine) and the negative charges on the exterior surface of tonoplast vesicles could change the coupling ratio of ATP hydrolysis to proton pumping. Thus, the surface charge of the tonoplast membrane may be involved in the regulation of these two activities.     

Purification of plasma membranes from dry maize embryos

Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficult. In the present work, plasma membranes were isolated from dry maize ( Zea mays L.) embryos with an enrichment of 11-fold as estimated by glucan synthase II (GSII, EC 2.4.1.34) activity and a purity of 78 to 90% as judged by the sensitivity of ATP hydrolysis to vanadate, a specific inhibitor of the plasma membrane H⁺-ATPase (EC 3.6.1.35). The procedure involved a double homogenization of the dry embryos and the addition of a 1500- g supernatant to an aqueous polyethyleneglycol-dextran two-phase partitioning system; the optimal ratio of polyethyleneglycol-dextran for purification of plasma membranes from dry seeds was 6.8/6.8% (w/w). In the isolated membranes a trace of a tonoplast enzyme marker (tonoplast H⁺-ATPase, EC 3.6.1.3) could be detected, but there were negligible amounts of mitochondrial and rough endoplasmic reticulum markers, H⁺-ATPase (EC 3.6.1.34) and diacylglycerol acyltrans-ferase (EC 2.3.1.20), respectively. The technique could also be used in hydrated embryos. The entire procedure can be carried out in 5 to 6 h. The resulting preparation is stable for at least 2 months at −70°C. The membranes of dry and hydrated embryos exhibited a high level of vanadate-sensitive ATPase activity that was increased by lysophosphatidylcholine.     

Phosphorylation-dephosphorylation of membrane proteins controls the microsomal H-ATPase activity of corn roots

The Mg²⁺-dependent H⁺-ATPase activity of a sealed microsomal vesicle fraction isolated from corn (Zea mays L.) roots appears to be controlled by a phosphorylation-dephosphorylation cycle. Phosphorylation of the microsomal fraction is carried out by a Ca²⁺/calmodulin (CaM)-stimulated process. The H⁺-ATPase activity decreases with increasing phosphorylation of the membranes and becomes only slightly uncoupled by ionophores and less inhibited by dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), NO₃⁻ and vanadate. The inhibitory effect of phosphorylation is greater on the NO₃⁻-sensitive H⁺-ATPase activity than on the vanadate-sensitive activity. Restoration of H⁺-ATPase activity is achieved by allowing the phosphorylated membranes to dephosphorylate either in the absence or presence of exogenous alkaline phosphatase. Moreover, the presence of fluphenazine during the Ca²⁺/CaM-stimulated treatment inhibits membrane phosphorylation and protects the H⁺-ATPase activity from inhibition.     

Cadmium induces acidosis in maize root cells

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.     

Characterization of a Ca2+-translocating ATPase from corn root microsomes

Brauer, D., Schubert C. and Tu, S,-I. 1990. Characterization of a Ca²⁺-translocating ATPase from corn root microsomes. - Physiol. Plant. 78: 335-344.
The existence of a Ca²⁺-translocating ATPase in microsomes from maize ( Zea mays L. cv, WF9 × Mo17) roots was evaluated using assays to follow Ca²⁺-stimulation of ATP hydrolysis and Ca²⁺ transport by changes in the fluorescence of chlorotetracycline, ATP hydrolysis by microsomes was stimulated by the addition of Ca²⁺ and further enhanced by the Ca ionophore A23187 and bovine brain calmodulin only in the presence of Ca²⁺, Stimulation by these agents was additive and sensitive to vanadate. These results were consistent with the presence of a Ca²⁺-translocating ATPase in microsomal membranes. The fluorescence of chlorotetracycline in the presence of microsomes and Ca²⁺ increased upon the addition of ATP, indicating the transport of Ca²⁺, The initial rate and extent of change in fluorescence were stimulated by calmodulin and quenched by the addition of either A23187 or EGTA, but not by protonophores. Changes in chlorotetracycline fluorescence were prevented by vanadate. Therefore, results using chlorotetracycline also indicated the presence of a Ca²⁺-translocating ATPase, Localization experiments indicated that the majority of the Ca²⁺-translocating ATPase was on the endoplasmic reticulum.     

Characterization of the pyrophosphate-dependent proton transport in microsomal membranes from maize roots

Cleared maize ( Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP- and pyrophosphate (PP_i)-dependent proton-pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PP_i-dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H⁺-PPase was Mg²⁺-dependent and the K_m for PP_i (in the presence of 3 m M MgSO₄) was 28 μ M . The pump was electrogenic, K⁺-dependent and a permeant anion was necessary to dissipate the membrane potential (NO₃⁻= I⁻ >Br⁻ > Cl⁻). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H⁺-PPase was insensitive to vanadate, oligomycin and molybdate. -Diethylstilbestrol (DES) and N,N'-dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μ M .     

Effects of dicyclohexylcarbodiimide (DCCD) treatment on coupled activities of vanadate-sensitive ATPase from plasma membrane of maize roots

The presence of dicyclohexylcarbodiimide (DCCD) inhibited the activities of vanadate-sensitive H⁺ -ATPase in both native and reconstituted plasma membrane of maize (Zea mays L. cv. WF9 × Mo 17) roots. Concentration dependence of DCCD inhibition on adenosine triphosphate (ATP) hydrolysis of native plasma membrane vesicles suggested that the molar ratio of effective DCCD binding to ATPase was close to 1. The DCCD inhibition of ATP hydrolysis could be slightly reduced by the addition of ATP, Mg:ATP, adenosine monophosphate (AMP), Mg:AMP and adenosine diphosphate (ADP). More hydrophilic derivatives of DCCD such as l-ethyl-N^?-3-trimethyl ammonium carbodiimide (EDAC) or 1-ethyl-3-3-dimethyl-aminopropyl carbodiimide (EDC) gave no inhibition, indicating that the effective DCCD binding site was located in a hydrophobic region of the protein. The proton transport activity of reconstituted plasma membrane at a temperature below 20°C or above 25°C was much sensitive to DCCD treatment. Build-up of the proton gradient was analyzed according to a kinetic model, which showed that proton leakage across de-energized reconstituted plasma membranes was not affected by DCCD, but was sensitive to the method employed to quench ATP hydrolysis. Reconstituted plasma membrane vesicles treated with DCCD exhibited a differential inhibition of the coupled H⁺-transport and ATP hydrolysis. The presence of 50 μM DCCD nearly abolished transport but inhibited less than 50% of ATP hydrolysis. The above results suggest that the link between proton transport and vanadate-sensitive ATP hydrolysis is indirect in nature.     

Calcium transport and ATPase activity in a microsomal vesicle fraction from corn roots

Abstract. An investigation has been made of methods for isolating membrane vesicles from corn ( Zea mays L.) roots active in calcium transport and K⁺-stimulated ATPase. Pretreating and grinding the roots at room temperature with EGTA and fusicoccin increases basal ATPase activity. Improvement in Ca²⁺ uptake requires isolation of a scaled vesicle fraction by the method of Sze(1980). Sorbitol is superior to sucrose as an osmoticant. The pH optimum for Ca²⁺ uptake is 7.5. whereas that for associated ATPase activity is 6.5. Calmodulin strongly stimulates Ca²⁺ uptake in a process little affected by uncouplers and ATPase inhibitors, but blocked by chlorpromazine. Fusicoccin gives less stimulation of Ca²⁺ uptake which is sensitive to uncouplers, and is dependent upon isolation with fusicoccin present. It appears that the sealed vesicle fraction may possess two Ca²⁺ transport systems: a calmodulin-activated Ca²⁺-transporting ATPase, and a Ca²⁺/H⁺ antiport coupled through the protonmotive force to a fusicoccin-stimulated H⁺-ATPase.     

Toxins of Pseudomonas syringae pv. syringae affect H+-transport across the plasma membrane of maize

The activity of some phytotoxic metabolites of Pseudomonas syringae pv. syringae Van Hall strains B359 and B301 on in vivo and in vitro systems of H⁺-transport across the plasma membrane of maize (Zea mays L., hybrid Paolo) was investigated. In particular syringomycin, the first lipodepsinonapeptide isolated from Pss and already studied in plants and yeasts for its effects on several physiological systems, was compared with the recently described lipodepsipeptides with 22 or 25 amino acid residues, so called syringopeptins. The in vivo activity of the phytotoxins was tested on fusicoccin-stimulated H^?-extrusion from cuttings of maize roots, which was inhibited by both types of toxins, with syringomycin more efficient than the syringopeptins. In vitro the H⁺-ATPase activity of predominantly right-side-out plasma membrane vesicles purified by two-phase partitioning was stimulated by 10 μM syringomycin and inhibited by higher levels, in agreement with the results of others with preparations of dicotyledons. Also the inhibition of the phosphohydrolytic activity of inside-out vesicles of mung bean plasma membrane was confirmed for maize. In both types of vesicles the syringopeptirts were better inhibitors than syringomycin. The pH gradient formed on addition of ATP to predominantly (25% latency) inside-out vesicles was immediately and completely collapsed by syringomycin and syringopeptins; H⁺-pumping was prevented if the toxins were added before ATP. The inhibition was concentration dependent, but at very low concentrations the effect was inverted. The results of the present investigation, carried out with maize preparations, confirm and extend the evidence so far obtained with dicotyledons in favour of the plasma membrane as an important site of interaction of syringomycin with the plant cell. They also indicate that, except for some details, the effects of syringopeptins at the level of the plasma membrane are the same as those of syringomycin.     

An ATP-driven proton pump in brush-border membranes from rat renal cortex

Summary The rate of ATP hydrolysis in ATP-preloaded plasma membrane vesicles derived from the luminal membrane of renal cortical tubules, and the rate of H⁺ secretion out of the same vesicles were investigated. Both were inhibited at low temperature, by the action of filipin, an antibiotic that complexes with cholesterol in plasma membranes, and by the action of blockers of mitochondrial F_o hydrogen channels, dicyclohexylcarbodiimide and Dio-9. Valinomycin in the presence of K⁺ showed a stimulatory effect, the protonophor carbonyl-cyanid-p-trifluormethoxy-phenylhydrazone stimulated the intravesicular ATP hydrolysis and apparently abolished acidification of the extravesccular medium. Lowering of the pH of the extravesicular medium retarded ATP hydrolysis, while readjustment of extra- and intravesicular pH accelerated ATP hydrolysis again. These findings strongly support the assumption that an ATP-driven proton pump is located in the luminal membrane of renal cortical tubules.     

Influence of K＋ on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H＋-ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean ( Glycine max L. ) hypocotyls by two-phase partitioning methods. The stimulatory effects of K+ on the coupling between ATP hydrolysis and proton transport by the plasma membrane H+-ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KC1, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that Km of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while Vmax of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg- l protein·min-1 in the presence of KC1. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KC1, respectively. Further studies revealed that K+ could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K+ could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H+ -ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H + -ATPase.     

Anion-sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë

Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G-25. The ATPase activity was dependent on the presence of divalent cations (Mg²⁺≥ Mn²⁺≥ Ca²⁺, Co²⁺; Zn²⁺ had no effect). Mg²⁺-dependent ATPase activity was stimulated by anions (Cl^? > malate²⁺, HCO^?₃), with maximal stimulation at concentrations above 50 mM. Mg²⁺-Dependent activity was inhibited by NO^?₃ above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K⁺ or Na⁺ was detected; stimulation by NH⁺₄ was abolished by 0.01% (w/v) Triton X-100, suggesting that the NH⁺₄ effect was due to the permeability of vacuolar membrane vesicles to NH₃. Trans-tonoplast electrical potentials (Δψ) and intra-vacuolar pH were measured with glass microelectrodes and antimony covered glass micro-pH-electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg-ATP, vacuoles showed an FCCP-sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x?± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg-ADP and ATP without Mg²⁺ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion-sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM.     

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

The effect of boron excess and deficiency on H⁺ efflux from excised roots from sunflower ( Heliarahus annuus L. cv. Enano) seedlings and on plasma membrane H⁺-ATPase (EC 3.6.1.35) in isolated KI-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H₃BO₃ (5 m M ) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H₃BO₃ concentration (0.25 m M ). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H⁺-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsorne vesicles. A strong effect of boron on the vanadate-sensitive, ATP-dependent H⁺ transport was found, but the vanadate-sensitive phospho-bydrolase activity was not affected. These results suggest that boron could exert an effect on the plasma membrane properties, directly or indirectly regulating, proton transport.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Effects of air pollutants on proton and sucrose transport at the plasma membrane of Ricinus communis

The effects of the air pollutants O₃, SO₂ and NO₂ on aspects of sucrose/proton cotransport across the plasma membrane of Ricinus communis plants have been investigated. The H⁺-ATPase hydrolytic activity in cotyledon plasma membrane vesicles purified by phase partitioning showed small stimulations by Na₂SO₃ or NaNO₃ added separately or together to the assay medium. ATPase activity from plants pretreated by fumigation with SO₂ or O₃ also showed an increase, the effect of O₃ being quite marked. Plasma membrane H⁺-pumping in KI-treated microsomal fractions and medium acidification by intact cotyledons both showed small decreases in the presence of Na₂SO₃ or NaNO₂. Both Na₂SO₃ and NaNO₂ at high concentrations (2 mol m^–3) had significant effects on sucrose uptake by intact cotyledons, although sucrose efflux was unaffected. No significant effects on sucrose uptake or efflux by intact cotyledons were observed in plants pretreated by fumigation with SO₂ or O₃. Proton-coupled sucrose transport in isolated plasma membrane vesicles was inhibited in the presence of Na₂SO₃ or NaNO₂. However, both pollutants also significantly inhibited the uptake of acetate by the vesicles, indicating a dissipation of the pH gradient across the membrane. It was concluded that no specific aspect of the sucrose/proton cotransport mechanism was damaged by these air pollutants, and that the effects of these pollutants on carbohydrate partitioning are more likely to be due to general effects on membrane integrity or on other aspects such as leaf carbohydrate metabolism.     

Characterization of reconstituted plasma membrane H+-ATPase from maize roots

Corn ( Zea mays L.) plasma membranes from KI-washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate-insensitive proton transport copurified with fractions containing vanadate-sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP-dependent H⁺-transport. Apparent K_i values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μ M , respectively; the KJ value of the H+-pumping of reconstituted ATPase was 30 μ M. The proton pumping of reconstituted vesicles could be discharged rapidly by p -trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg-ATP by both native and reconstituted ATPases obeyed simple Michaelis-Menten plots with a K_m between 0.5 and 0.6 m M. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.     

玉米重金属铅(Pb~(2+))含量的配合力分析与育种对策

选择有代表性的12个玉米自交系,按Griffing4模式组配获得66个组合(F1),用温室盆栽,在3个Pb2+污染水平下对叶片和子粒Pb2+含量配合力和遗传参数进行分析。结果表明:玉米叶片和子粒的Pb2+含量一般配合力与特殊配合力差异均达到显著水平,非加性方差大于加性方差,遗传方差大于环境方差,广义遗传率大于狭义遗传率,该性状的变异主要来自遗传因素,遗传力较强。玉米种质筛选过程中,土壤Pb2+浓度在333.32 mg/kg以下,用亲本郑58组配的组合在筛选时不仅注重子粒Pb2+含量未超标而且要注重叶片Pb2+高富集,其主要是兼顾饲料和粮食安全的同时进行土壤Pb2+污染的生物修复;土壤Pb2+浓度高于715.46 mg/kg时,用亲本178组配的组合筛选应注意叶片和子粒低Pb2+积累的种质选育,对今后在不同Pb2+污染土壤中开展玉米品种筛选和规避污染育种策略的选择具有一定的指导意义。     

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H⁺ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H⁺ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H⁺ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H⁺ reverses the direction of the H⁺ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H⁺ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.