ATP stimulates the uptake of S-dinitrophenylglutathione by rat liver plasma membrane vesicles

Incubation of inverted plasma membrane vesicles from rat liver with micromolar concentrations of S-dinitrophenylglutathione (DNP-SG) in the presence of ATP resulted in the uptake of DNP-SG into the vesicles. ATP-dependent DNP-SG accumulation was half-maximal with 9 μM DNP-SG, while the K_m for ATP was 320 μM. Glutathione disulfide (GSSG), but not reduced glutathione, inhibited the ATP-dependent accumulation of DNP-SG by the vesicles, suggesting that the same, ATP-dependent transport system is responsible for the extrusion of glutathione conjugates and GSSG from liver cells.     

Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

A fluorescent compound in oat root plasma membrane

Homogenates of 7-day-old oat (Avena sativa L. cv. Brighton) roots were highly fluorescent (excitation and emission maxima around 360 and 440 nm, respectively). Less than ¹/₁₀ as much fluorescence per g fresh weight was found in oat shoots or in wheat (Triticum aestivum L. cv. Drabant) roots or shoots. Most of the fluorescence of oat roots was found in the soluble fraction (150 000g supernatant). However, some could be detected in the plasma membrane fraction (excitation and emission maxima 365 and 417 nm, respectively), which contained a 3-fold higher fluorescence per mg protein than the homogenate. Growth of oat or wheat in a medium containing, 10-^?5M scopoletin (6-methoxy-7-hy-droxy coumarin), a fluorescent compound previously reported to be present in both wheat and oat roots, caused the disappearance of scopoletin from the medium (proportional to the amount of roots) and the appearance of increased fluorescence in the root homogenates but not in the shoot homogenates. In both oat and wheat roots ail of the extra fluorescence was recovered in the soluble fraction and at least in wheat it consisted of unconverted scopoletin. The concentration of scopoletin in wheat roots grown in 10-^?5M scopoletin was around 50 nmol (g fresh weight)^?1, or about five times the concentration in the growth medium. Scopoletin in the growth medium (10-^?5M) or in the assays (up to 10-^?4M) did not affect Mg²⁺-, Mg²⁺+K⁺- or Ca²⁺-ATPase activities in wheat or oat roots. The fluorescence properties of the oat plasma membrane were different from those of authentic scopoletin. Either the surroundings modify the fluorescence of membrane-associated scopoletin or the endogenous fluorescent compound is not scopoletin but a glycoside-derivative of scopoletin or some completely unrelated compound.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Early Sr2+-induced effects on membrane potential,proton pumping- and ATP hydrolysis-activities of plasma membrane vesicles from maize root cells

When released in plant environment, strontium (Sr²⁺) can be absorbed predominantly by the plant roots. As the plasma membrane of root cells is amongst the first barriers encountered by Sr²⁺ during its soil/plant transfer and the main entry point of Sr²⁺ into the roots, the main objective of this work aimed to enlighten on some of the Sr²⁺-induced effects at this level in Zea mays L. cv. “Liberal”.Thus this study focused on the Sr²⁺-induced changes on membrane potential of cortical root cells and on proton fluxes in maize roots, in order to determine whether the activity of some of the ion transport systems present in the plasma membrane of maize root cell could be among the first targets of Sr²⁺. We focused in particular on the plasma membrane H⁺-ATPase, known to be one of the major transport systems found in the plasmalemma where it generates a proton motive force (contributing to membrane potential maintaining, and providing energy for ion transport through membrane).The data presented here showed that Sr²⁺ triggered an early and transient membrane depolarisation whose magnitude and duration were dependent on the Sr²⁺-concentration. The time course pattern of a second longer lasting depolarisation could be examined in perspective with the Sr²⁺-induced decrease of the spontaneous proton extrusion observed in root tissues, suggesting a relationship between Sr²⁺-effects on membrane potential and H⁺ excretion. Furthermore, the inhibitory effect exerted by Sr²⁺ on the fusicoccin (FC)-enhanced proton extrusion strongly suggested an inhibition of the plasma membrane H⁺-ATPase. This hypothesis was supported by the inhibition induced by Sr²⁺ on proton pumping- and ATP hydrolysis-activities measured in plasma membrane vesicles (PMV) prepared from maize roots.Taken together the data reported here evidence that, with however a lower efficiency, Sr²⁺ behaved in a quite similar way to Ca²⁺ when inhibiting the H⁺-ATPase activity, and suggest that Sr²⁺ could partially mimic Ca²⁺ onto regulation of the H⁺-ATPase activity.     

Lysophosphatidylcholine specifically stimulates plasma membrane H(+)-ATPase from corn roots

The plasma membrane H(+)-ATPase activity from corn seedling roots is shown to be stimulated 3- to 4-fold by the addition of lysophosphatidylcholine (lysoPC). This effect clearly differs from that of other detergents by both the magnitude and the absence of inhibition at higher concentrations. LysoPC decreases the apparent Km for MgATP, increases Vmax of the ATPase reaction but does not change its pH optimum. On the contrary, the acid phosphatase activity associated with plasma membranes is not influenced by lysoPC. A lysoPC stimulation is also demonstrated for the solubilized preparation of the H(+)-ATPase. It is assumed that lysoPC stimulation of the plant plasma membrane H(+)-ATPase is not only due to permeabilization of the vesicles for MgATP, but also to direct action on the enzyme.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Characterization of passive ion transport in plasma membrane vesicles of oat roots

The passive influx and efflux of inorganic ions across plasma membrane vesicles purified from extracts of Avena sativa roots were investigated. Uptake was measured by incubating the vesicles in a radioisotope for various times. The “loaded” vesicles were separated from the external solution by gel filtration. Efflux was measured by dialyzing the preloaded vesicles.     

Characterization of a glutamine/proton cotransporter from Ricinus comnmnis roots using isolated plasma membrane vesicles

The mechanism of glutamine transport at the plasma membrane of sink tissue cells was investigated using isolated plasma membrane vesicles from roots of Ricinus communis L. var. sanguineous . Glutamine transport was found to be driven by both the pH gradient (ΔpH) and a membrane potential (ΔΨ) (alkaline and negative internal), which were created artificially across the plasma membrane. Glutamine wus accumulated 15–20-fold in the presence of both a ΔpH and Δ Ψ . There appeared to be a direct pH effect on Δ PS -driven transport, as a higher rate of transport was observed at pH 5.5 than at pH 7.5. The ΔpH +Δ Ψ -driven transport showed saturation kinetics with a K_m of 287 μ M . Altering the membrane potential changed the V_max but had no effect on the K_m of glutamine transport. These results are consistent with the presence of a proton-coupled, carrier-mediated system for glutamine uptake in Ricinus roots. A range of protein modifiers and transport inhibitors had limited effects on glutamine transport: highest inhibition uas observed with cytochalasin D. When glutamine transport was compared in plasma membrane vesicles isolated from the root lips of Ricinus and from the remainder of the root tissue a lower level of transport was observed in the root tips. A method for the solubilization and reconstitution of glutamine transport activity using the detergent CHAPS is also described.     

Calmodulin dependent formation of membrane potential in barley root plasma membrane vesicles: A biochemical model of aluminum toxicity in plants

Micromolar concentrations of aluminum ions interfere with calmodulin-stimulated, membrane bound ATPase activity which plays a role in the maintenance of the transmembrane potential of plasma membrane enriched vesicles isolated from barley roots. Calmodulin appears to be the major target for aluminum interaction resulting in pronounced changes in the exposure of a large, hydrophobic surface on this protein as determined with a fluorescent, hydrophobic surface probe. At a molar ratio of 3:1 [aluminum]/[calmodulin], the calmodulin stimulated enzymatic activity, probably associated with a Ca²⁺+ Mg²⁺ATPase, is about 95% inhibited. Aluminum induced changes in calmodulin structure are reflected in reduced formation of the membrane potential when assayed with a fluorescent potential probe, oxonol VI. We hypothesize that the aluminum caimodulin complex represents a primary lesion in toxic responses of plants to this metal.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

ATP synthesis by an artificial proton gradient in right-side-out membrane vesicles of Escherichia coli.

Membrane vesicles formed from spheroplasts of E. coli lysed in the presence of ADP and P_i produced ATP when an artificial proton gradient (acid outside) was formed across the membrane. ATP synthesis required Mg²⁺ and ADP, was inhibited by dicyclohyxylcarbodiimide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by valinomycin in the presence of KCl. Synthesis was absent in a mutant lacking the Mg²⁺-ATPase. The optimum external pH was 2.5 when the internal pH was 8.2. Oxidative phosphorylation driven by D-lactate or succinate was also observed.     

Glucose transport activity in isolated plasma membrane vesicles from Saccharomyces cerevisiae

Purified plasma membranes prepared from yeast cells by mechanical agitation with glass beads exhibit no detectable sugar transport activity. However, the addition of phospholipid (asolectin) liposomes to the purified plasma membranes followed by freezing, thawing, and brief sonication produces membrane vesicles which exhibit D-glucose-specific transport activity. The characteristics of zero trans, equilibrium exchange, and influx counterflow exhibited by the membrane vesicles are similar to those of intact cells.     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 M-1 min-1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.     

Cl- transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

Gradually altered synthetic entities were employed as molecular probes, and arachidonic acid, ADP, human alpha-thrombin and the Ca2+ ionophore A23187 as aggregation-inducing agents, in a comprehensive study on the response profile of human blood platelets with an emphasis on the effects of exogenous and increased intracellular Ca2+. Corroborating further previous conclusions, some representative carbamoylpiperidine derivatives, at concentrations effecting substantial inhibition of ADP-induced aggregation, failed to retain that effect when 5.0 mM Ca2+ was introduced into the otherwise identical test medium; reference compounds chlorpromazine and propranolol registered corresponding inhibitory patterns. At increased concentrations the compounds' inhibitory potency was regenerated even in the presence of 5 mM Ca2+. In fact, in sufficiently high concentrations, the compounds were even capable of inhibiting aggregation elicited by 15 microM of the ionophore A23187; so did chlorpromazine and propranolol. Another set of congeners revealed the striking sensitivity of ionophore A23187-induced human blood platelet aggregation to the surface active potencies of inhibitor molecules. The loss in inhibitory potency was directly related to the lesser hydrophobic character of the molecule.     

Characterization of DIDS-sensitive ATP accumulation in brain synaptic vesicles

ATP is an excitatory neurotransmitter in the central and peripheral nervous system. We investigated ATP accumulation in highly purified brain synaptic vesicles (SVs). Based on the amount of ATP accumulated in SVs under the conditions used, ATP is not transported against a concentration gradient but rather appears to have a Delta mu H(+)-independent mechanism. ATP transport was inhibited by DIDS and NEM, but was not affected by Mg(2+) or by pre-incubation with nucleotides. These results suggest a unique transport mechanism that does not involve exchange with other nucleotides or protons, unlike other known neurotransmitter transport systems.     

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.