Plasma Membrane-enriched Vesicles Research Articles

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC3), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC3 to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC3 or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC3 and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC 50 of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC3 to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC3/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC3 and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.

Changes in H+-ATPase activity of plasma membrane-enriched vesicles isolated from physic nut roots and leaves of energy and medicinal plant, Jatropha curcas L., as an adaptation mechanism to salinity

The plasma membrane H+-ATPase (PM H+-ATPase) plays a key role in the activation of ion and nutrient transport, and is proposed to be involved in salt tolerance. This study investigates the acclimation of PM H+-ATPase in physic nut (Jatropha curcas L.) roots and leaves treated with 0, 50 and 200 mM NaCl for 5 days. Upon comparison with control roots, the H+-ATPase hydrolytic activity, Vmax, Km, H+-pumping activity and pH gradient potential across the plasma membrane were significantly higher in roots treated with NaCl, especially under mild salt stress. The translational activation of PM H+-ATPase of physic nut roots helped to maintain K+ and Ca2+ uptake as well as nitrogen and phosphorus uptake in roots. Compared with the control leaves, the maintenance of H+ transport under 50 mM NaCl was attributed to a modification of the lipid membrane, and the increase in H+transport under 200 mM NaCl could be because of an acid-mediated activation. Differential expression of the H+-ATPase isoforms could also occur in leaves under salt stress. Thus, the stable pH gradient created by the H+-ATPase in leaves treated with 50 mM NaCl assisted normal leaf growth. These modulations of PM H+-ATPase in the roots and leaves of physic nuts could represent a set of adaptive mechanisms to salinity. Key words: Immunoprecipitation, Jatropha curcas L., plasma membrane H+-ATPase, salt stress.

Changes in growth irradiance are reflected on H +ATPase activity of plasma membrane enriched vesicles from maize ( Zea mays L.) roots

Leaves change their photosynthetic activity in response to growth light conditions, but little is known about what may happen at the root level. The effect of irradiance level (high or low) on transport activities of root plasma membrane enriched vesicles was studied in maize ( Zea mays L.) plants. High irradiance appears to have a differential promoting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H +-accumulation. Furthermore, our results put in evidence a correlation between increase in enzyme activity and increase in MHA2 gene transcription level. Finally, high irradiance results in increased uptake rates of nitrate and in a higher reduction rate of the anion. We suggest that high light-induced changes in plasma membrane H +ATPase activity and transcription might have an adaptive role in sustaining the higher request for the nitrate resulting from increased photosynthate availability.

Calcium Inhibits Dihydropyridine-Stimulated Increases in Opening and Unitary Conductance of a Plant Ca2+ Channel

We have previously characterized the "RCA" channel (root Ca²+ channel), a voltage-dependent, Ca²+-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers. Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202-791). However, the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca²+, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca²+ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202-791 or nifedipine caused an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive interaction between Ca²+ and DHPs at one of the Ca²+-binding sites involved in Ca²+ permeation and that binding of a DHP to one of the Ca²+-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant Ca²+-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between plant and animal Ca²+-permeable channels.

Responses of two ecotypes of Medicago ciliaris to direct and bicarbonate-induced iron deficiency conditions

Our study investigates the effect of iron deficiency on morpho-physiological and biochemical parameters of two Medicago ciliaris ecotypes (Mateur TN11.11 and Soliman TN8.7). Iron deficiency was imposed by making plants grow, either in an iron free or by the addition of CaCO3/NaHCO3 to the Hoagland nutrient solution. Our results showed that both true and bicarbonate Fe-deficiency induced the characteristic iron-chlorosis symptoms, although the intensity of the symptoms was ecotype-dependent. This variability in tolerance to iron deficiency was also displayed by other morphological parameters such as root biomass and chlorophyll concentration. Besides, iron chlorosis induced an increase in biochemical parameters: the iron reducing capacity (measured in vivo on root segments and in vitro on plasma membrane enriched vesicles) and rhizosphere acidification by enhancement of H+-ATPase activity were more pronounced in Mateur ecotype. These findings suggest that Soliman ecotype was more sensitive than Mateur one to iron chlorosis.

Signaling from soybean roots to Rhizobium

Legume plants have a unique ability to fix atmospheric nitrogen via symbiosis with rhizobia. For the establishment of symbiosis, legume plants secrete signaling molecules such as flavonoids from root tissues, leading to the attraction of rhizobia and the induction of rhizobial nod genes. Genistein and daidzein are found in soybean root exudates and function as signal molecules in soybean – Bradyrhizobium japonicum chemical communication. Although it is more than 20 years since these signal flavonoids were identified, almost nothing has been characterized concerning the membrane transport process of these molecules from soybean roots. To elucidate the transport mechanism we performed membrane transport assays with plasma membrane-enriched vesicles and various inhibitors. As a result, we concluded that an ATP-binding cassette-type transporter is involved in the secretion of genistein from soybean roots. The possible involvement of a pleiotropic drug resistance-type ABC transporter in this secretion is also discussed.

A novel method to quantify H +-ATPase-dependent Na + transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na + is excluded from the cytosol to the apoplast or the vacuole by Na +/H + antiporters. The secondary active transport of Na + to apoplast against its electrochemical gradient is driven by plasma membrane H +-ATPases that hydrolyze ATP and pump H + across the plasma membrane. Current methods to determine Na + flux rely either on the use of Na-isotopes ( 22Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H + gradient due to H +-ATPase in the presence or absence of Na + by pH-sensitive probes. To date, there are no methods that can directly quantify H +-ATPase-dependent Na + transport in plasma membrane vesicles. We developed a method to measure bidirectional H +-ATPase-dependent Na + transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H +-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na + sealed for the study of H +-ATPase-dependent Na + transport. Our data implicate that Na + movement across vesicle membranes is highly dependent on H +-ATPase activity requiring ATP and Mg 2+ and displays optimum rates of 2.50 μM Na + mg − 1 membrane protein min − 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H +-ATPase-dependent Na + transport. The results demonstrate that the Na + content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H +-ATPase-dependent Na + transport with membrane vesicles.

Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells

SPICK2, a homologue of the weakly-inward-rectifying Shaker-like Arabidopsis K channel, AKT2, is a candidate K+-influx channel participating in light- and clock-regulated leaf movements of the legume, Samanea saman. Light and the biological clock regulate the in situ K+-influx channel activity differentially in extensor and flexor halves of the pulvinus (the S. saman leaf motor organ), and also-though differently-the transcript level of SPICK2 in the pulvinus. This disparity between the in situ channel activity versus its candidate transcript, along with the sequence analysis of SPICK2, suggest an in situ regulation of the activity of SPICK2, possibly by phosphorylation and/or by interaction with cAMP. Consistent with this (i) the activity of the voltage-dependent K+-selective fraction of the inward current in extensor and flexor cells was affected differentially in whole-cell patch-clamp assays promoting phosphorylation (using the protein phosphatase inhibitor okadaic acid); (ii) several proteins in isolated plasma membrane-enriched vesicles of the motor cells underwent phosphorylation without an added kinase in conditions similar to patch-clamp; and (iii) the SPICK2 protein was phosphorylated in vitro by the catalytic subunit of the broad-range cAMP-dependent protein kinase. All of these results are consistent with the notion that SPICK2 is the K+-influx channel, and is regulated in vivo directly by phosphorylation.

In vivo and in vitro effects of cadmium on H +ATPase activity of plasma membrane vesicles from oat ( Avena sativa L.) roots

The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons.

Response of the charophyte Nitellopsis obtusa to heavy metals at the cellular, cell membrane, and enzyme levels.

The responses of the freshwater macroalga Nitellopsis obtusa to heavy metal (HM) salts of Hg, Cd, Co, Cu, Cr, and Ni were assessed at different levels: whole-cell mortality (96-h LC(50)), in vivo cell membrane (45-min depolarization of resting potential, EC(50)), and enzyme in plasma membrane preparations (K+, Mg2+-specific H+-ATPase inhibition, IC(50)). To measure ATPase activity, a novel procedure for isolation of plasma membrane-enriched vesicles from charophyte cells was developed. The short-term ATPase inhibition assay (IC(50) from 6.0 x 10(-7) to 4.6 x 10(-4) M) was slightly more sensitive than the cell mortality test (LC(50) from 1.1 x 10(-6) to 2.6 x 10(-3) M), and the electrophysiological test with the end point of 45-min depolarization of resting potential was characterized by less sensitivity for HMs (EC(50) from 1.1 x 10(-4) to 2.2 x 10(-2) M). The variability of IC(50) values assessed for HMs in the ATPase assays was close to that of LC(50) values in the mortality tests (CVs from 33.5 to 83.5 and from 12.4% to 57.7%, respectively), whereas the EC(50) values in the electrophysiological tests were characterized by CVs generally below 30%. All three end points identified two separate HM groups according to their toxicity to N. obtusa: Co, Ni, and Cr comprised a group of less toxic metals, whereas Hg, Cu, and Cd comprised a group of more toxic metals. However, the adverse effects within each group were discriminated differently. For example, the maximum difference between the highest and lowest LC(50) for the group of less toxic metals in the long-term mortality test was approximately 60% of the response range, whereas the corresponding difference in IC(50) values in the ATPase assay was 30%. In contrast, the LC(50) values of the more toxic metals occupied only 10% of the response range, whereas the IC(50) values were spread over 70%. Further investigation should be done of the underlying mechanism or mechanisms responsible for the observed differences in the dynamic range of a particular end point of the groups of toxicants of varying strength.

Tolerance to NaCl induces changes in plasma membrane lipid composition, fluidity and H+-ATPase activity of tomato calli.

Two tomato (Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 mM NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H+-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 mM NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 mM NaCl, growth of calli tolerant to 100 mM NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 mM NaCl. Growth in the presence of 100 mM NaCl provoked an increase of Na+ and Cl- content, but no significant changes in K+ and Ca2+. As compared with NaCl-sensitive and 50 mM NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 mM NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 mM NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 mM NaCl showed lower ATP hydrolysis and ATP-dependent H+-pumping activities, as well as a lower passive permeability to H+ than plasma membrane from NaCl-sensitive and 50 mM NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H+-ATPase activity in salt tolerance of tomato calli is discussed.

Single-channel characterization of a nonselective cation channel from human placental microvillus membranes. Large conductance, multiplicity of conductance states, and inhibition by lanthanides.

The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl- channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P(Cl)/P(K) approximately or approximately equal 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a "promiscuous" connectivity pattern. The occurrence of both "staircaselike" and "all-or-nothing" transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation.

Effects of salt stress on H +-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots

It has previously been shown that mild and severe salt-stress increased both ATP- and PP i-dependent H + transport and induced a Na +/H + exchange activity in tonoplast vesicles from sunflower seedling roots. In this study, the vanadate-sensitive H +-ATPase activity was characterized in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to 0, 75 or 150 mM NaCl. Blots immunoassayed with an Arabidopsis thaliana plasma membrane ATPase polyclonal antibody revealed the existence of a band of about 100 kDa, which was highly enriched in the 32/43% sucrose interface. ATP hydrolyzing activity in this fraction was mostly inhibited by vanadate, and scarcely by azide, oligomycin, nitrate and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Properties of vanadate-sensitive ATPase activity, such as inhibitor sensitivity, pH optimum, substrate specificity, ion effects and kinetic data were consistent with those of other plasma membrane ATPases. Although the pH profile, ion dependence, apparent K m and the amount of antigenic protein were unaffected by salt-stress, the V max of the vanadate-sensitive ATPase activity (measured in the presence of detergent) was reduced as a function of salt treatments. Likewise, while mild and severe salt-stress reduced the basal ATP hydrolyzing activity (measured in the absence of detergent) about a 35 and 55%, respectively, the ATP-dependent H + transport activity sensitive to vanadate remained unchanged. As a consequence, the ratio of H + pumping to the basal phosphohydrolase activity was proportionally increased. These results and those previously reported at the sunflower tonoplast suggest that the activity of H + pumps under salt stress can form part of a tolerance mechanism in sunflower roots, which could regulate the ion fluxes (Na + and Cl −) across the tonoplast and plasma membrane.

Properties of two multisubstate Cl- channels from human syncytiotrophoblast reconstituted on planar lipid bilayers.

We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation, and Mg2+-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated in most cases, a result consistent with the higher permeability for cations as compared with Cl- and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode techniques. In this paper, we report that Cl--selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role of the reported Cl- channels in transplacental ion transport and/or syncytium homeostasis remains to be determined.

Calcium channels in higher plant cells: selectivity, regulation and pharmacology

Rapid influx of Ca(2+) into the cytosol from extracellular pools or intracellular stores via ion channels can have wide-ranging physiological consequences. In addition, influx of Ca(2+) across the plasma membrane is necessary for the large net accumulation of Ca(2+) essential for cellular integrity. In this paper, the properties of Ca(2+) channels in various plant membranes are reviewed, and compared with new results on the Ca(2+) channel from the plasma membrane of wheat roots (rca channel) described originally by Piñeros and Tester (1995). The rca channel has been studied at the single channel level by incorporation of plasma membrane-enriched vesicles into planar lipid bilayers. It has a high affinity for Ca(2+) permeation (K(m) = 99 µM) and a maximal conductance of 30 pS. It is highly selective for Ca(2+) over Cl(-), but allows the movement both of other divalent cations (with a conductivity sequence: Ba(2+) > Sr(2+) > Ca(2+) >Mg(2+) > Mn(2+)) and of monovalent cations. The affinity for K(+) permeation was 6 mM, and the maximal conductance was 164 pS. The permeability ratio, P(Ca(2+))/P(K(+)) ranged from 17 to 41, decreasing with increasing extracellular Ca(2+). With physiologically reasonable membrane potentials and ionic conditions, the channel will catalyse Ca(2+) influx. At normal resting potentials (negative of about -135 mV) the channel remains largely closed, but activates rapidly upon depolarization. It is insensitive to ABA and Ins 1,4,5-P(3), but the voltage-dependence for activation was shifted to more negative potentials upon addition of cytosolic ATP. The channel was inhibited by a range of trivalent cations (La(3+), Al(3+) and Gd(3+)) and by some organic Ca2+ channel effectors (verapamil, diltiazem, ruthenium red), although it was insensitive to bepridil and 1,4 dihydropyridines [nifedipine and (+) and (-) 202-791], at least in the conditions described here. The properties of this channel are compared with those of other plant and animal Ca(2+) channels, and are shown to be consistent with its proposed physiological role of divalent cation uptake into roots.

Trypanothione overproduction and resistance to antimonials and arsenicals in Leishmania.

Leishmania resistant to arsenicals and antimonials extrude arsenite. Previous results of arsenite uptake into plasma membrane-enriched vesicles suggested that the transported species is a thiol adduct of arsenite. In this paper, we demonstrate that promastigotes of arsenite-resistant Leishmania tarentolae have increased levels of intracellular thiols. High-pressure liquid chromatography of the total thiols showed that a single peak of material was elevated almost 40-fold. The major species in this peak was identified by matrix-assisted laser desorption/ionization mass spectrometry as N1,N8-bis-(glutathionyl)spermidine (trypanothione). The trypanothione adduct of arsenite was effectively transported by the As-thiol pump. No difference in pump activity was observed in wild type and mutants. A model for drug resistance is proposed in which Sb(V)/As(V)-containing compounds, including the antileishmanial drug Pentostam, are reduced intracellularly to Sb(III)/As(III), conjugated to trypanothione, and extruded by the As-thiol pump. The rate-limiting step in resistance is proposed to be formation of the metalloid-thiol pump substrates, so that increased synthesis of trypanothione produces resistance. Increased synthesis of the substrate rather than an increase in the number of pump molecules is a novel mechanism for drug resistance.

Plasma Membrane H+-ATPase Activity in Spores, Germ Tubes, and Haustoria of the Rust FungusUromyces viciae-fabae

Using plasma membrane-enriched vesicles, the properties of the H+-ATPase (EC 3.6.1.35) from the rust fungusUromyces viciae-fabaewere studied. The enzyme is strictly Mg2+-dependent and is inhibited by vanadate. The pH-optimum is at 6.7. By Western blot analysis using a monoclonal antibody against corn plasma membrane H+-ATPase a polypeptide of approximately 104 kDa could be detected. The vanadate-sensitive H+-ATPase activity of microsomal vesicles obtained from different stages of rust development was determined. Uredospores had only a very low enzyme activity (1.9 μmol Pi × mg−1protein × h−1). In germ tubes the ATPase activity was about twofold higher (4.0 μmol Pi × mg−1protein × h−1). An eightfold higher ATPase activity (16.1 μmol Pi × mg−1protein × h−1) was found in microsomal vesicles from haustoria which had been isolated from rust-infectedVicia fabaleaves. These results suggest, that the electrochemical gradient generated by the H+-ATPase of haustoria plays an important role for their function, possibly by promoting nutrient uptake from host cells.

Changes in Plasma Membrane H +-ATPase Activity during Aeration of Maize Roots

Summary Plasma membrane-enriched vesicles were isolated by density gradient centrifugation from maize roots previously submersed for 2 h with or without aeration in a 1 mmol/L CaS0 4 solution, a condition which can modify the rate of ion fluxes in root tissue. The treatments did not significantly modify the composition of the membrane fraction collected at the sucrose gradient interface, as revealed by marker enzyme activities. However, submersion of maize roots for 2 h in aerated CaS0 4 before membrane isolation caused a significant increase in both the initial rate of ATP-dependent proton pumping (+99 %) and the rate of ATP hydrolysis (+64 %) as compared with the vesicles from roots immediately homogenised after being removed from the seedlings. Simple submersion of maize roots without aeration of the solution induced only a small effect on both ATP-hydrolytic activity (+14 %) and ATP-dependent H + transport (-16 %). Submersion of root tissue clearly enhanced the apparent passive permeability of isolated membrane vesicles to protons irrespective of the aeration treatment. These results suggest the involvement of plasma membrane H + -ATPase in the response to changes in root environmental conditions, like submersion with or without aeration.

Modification of Proton Transport Kinetics of the Plasma Membrane H +-ATPase after Adaptation of Tobacco Cells to NaCI

Summary Plasma membrane enriched vesicle fractions were isolated on discontinuous sucrose gradients from unadapted and 428 mM NaCl adapted tobacco cells ( Nicotiana tabacum L. var. Wisconsin 38). The fractions collected from the 36/45 % interfaces were determined to be plasma membrane enriched based on analyses of marker enzyme activities. Kinetic properties of H + -transport (determined by [ 14 C]-SCN - uptake) of the plasma membrane H + -ATPase of adapted cells were substantially different than those of unadapted cells. H + transport activity of the plasma membrane H + -ATPase from NaCl adapted cells was more susceptible to substrate inhibition by free ATP (k i = 3.1 mM) than the activity of the plasma membrane H + -ATPase from unadapted cells (k i = 10 mM). The plasma membrane H + -ATPase from NaCl adapted cells exhibited an apparent K m for MgATP of 0.6 ± 0.2 mM compared with 1.76 ± 0.5 mM for the plasma membrane H + -ATPase from unadapted cells. These data indicate that the plasma membrane H + -ATPase of adapted cells can function efficiently in saline environments where increased competition for the ATP pool is presumed.

Effect of Zinc Deficiency on Proton Fluxes in Plasma Membrane-Enriched Vesicles Isolated from Bean Roots

Plasma membrane-enriched vesicles were isolated by density gradient centrifugation from roots of zinc-sufficient and zinc-deficient bean (Phaseolus vulgaris L. cv. Prelude) plants. The two populations of vesicles had similar activities of specific membrane marker enzymes and ATP hydrolysis and were competent for proton transport. However, vesicles from zinc-deficient roots showed lower rates of ATP-dependent intravesicular acidification and increases in passive permeability to protons as well as in the rate of dissipation of a non-metabolic transmembrane pH gradient. The decrease of the rate of proton accumulation in isolated vesicles closely paralleled the increase in potassium leakage from intact roots and the appearance of visual zinc deficiency symptoms in the shoots. Re-supply of zinc to deficient plants for 24 h promoted shoot growth, reduced potassium leakage from roots and led to partial recovery of the proton accumulation capacity and to a decrease in passive permeability to protons in isolated vesicles. The results obtained with isolated vesicles confirm the previously observed 'in vivo' effects of zinc deficiency and are consistent with the idea that an alteration of plasma membrane lipids leads to an increase in permeability and an impairment in trans plasma membrane proton gradient.