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1.
Kobara A Hiasa M Matsumoto T Otsuka M Omote H Moriyama Y 《Archives of biochemistry and biophysics》2008,469(2):195-199
Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters. 相似文献
2.
Hiasa M Matsumoto T Komatsu T Omote H Moriyama Y 《American journal of physiology. Cell physiology》2007,293(5):C1437-C1444
Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H(+)-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka et al. Proc Natl Acad Sci USA 102: 17923-17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587-593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H(+)-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K(+). Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H(+)/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations. 相似文献
3.
Komatsu T Hiasa M Miyaji T Kanamoto T Matsumoto T Otsuka M Moriyama Y Omote H 《The international journal of biochemistry & cell biology》2011,43(6):913-918
Human multidrug and toxic compound extrusion 2 (hMATE2) is a kidney-specific isoform of hMATE1, an exporter of toxic organic cations (OCs) of exogenous and endogenous origins at the final excretion step in the kidneys and liver (Otsuka et al., 2005), and contains a splicing variant, MATE2K, that has an exon of hMATE2 deleted (Masuda et al., 2006). In the present study, we characterized the degree of expression and the transport properties of hMATE2. Quantitative PCR analysis with probes specific for hMATE2 indicated the presence of hMATE2 mRNA in the kidneys, which corresponded to 39% of total mRNA encoding both hMATE2 and hMATE2K. hMATE2-specific antibodies immunostained the renal urinary tubules. Upon expression in HEK293 cells, hMATE2 was localized in intracellular vesicular structures, and thus transport activity of tetraethylammonium (TEA), a typical substrate for MATE transporters, by the cells was not detected. The hMATE2 protein was purified and reconstituted into liposomes. An artificially imposed pH gradient (ΔpH) across the proteoliposomal membrane drove the uptake of TEA. Dissipation of ΔpH by ammonium sulfate effectively inhibited the TEA uptake, while that of the membrane potential by valinomycin had little effect. The profiles of cis-inhibition of TEA transport by hMATE2 and hMATE2K are similar to each other. Thus, both hMATE2 and hMATE2K equally operate in the human kidneys to extrude OCs into the urine. 相似文献
4.
《生物化学与生物物理学报:生物膜》2018,1860(11):2456-2464
Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H+/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H+ exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport. 相似文献
5.
Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H(+)) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1-mediated efflux of the prototypic MATE1 substrate, [(3)H]1-methyl-4-phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC(50)/K(i) for interaction between extracellular H(+) and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different (P = 0.6). Furthermore, kinetics of interaction between intracellular H(+) and inward-facing hMATE1 determined using the efflux protocol revealed an IC(50) for H(+) of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H(+) with the inward-facing and outward-facing aspects of hMATE1. 相似文献
6.
MARRE M. T.; ALBERGONI F. G.; MORONI A.; MARRE E. 《Journal of experimental botany》1989,40(3):343-352
In Elodea densa leaves light strongly stimulates electrogenic,K +-dependent, vanadate- and erythrosin B-sensitive H+ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H+ extrusion in thedark. Light-induced H+ extrusion requires the presence of K+in the medium and is associated with increased K+ uptake andalkalinization of the cell sap. Light-induced H+ extrusion increaseswith increased CO2 concentration. At constant CO2 concentration(104 parts 106) the rate of H+ extrusion is stronglyenhanced by an increased light intensity up to 30 W m2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H+ extrusion, K+ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H+ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H+-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H+-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H+ extrusionand of K+ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H+ pump, K+ uptake, Elodea densa 相似文献
7.
Wataru Nishima Wataru Mizukami Yoshiki Tanaka Ryuichiro Ishitani Osamu Nureki Yuji Sugita 《Biophysical journal》2016,110(6):1346-1354
Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses H+ to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant H+-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another H+ from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic H+-coupled MATE multidrug transporters with the relevant aspartic acids. 相似文献
8.
Amlal Hassane; Wang Zhaohui; Soleimani Manoocher 《American journal of physiology. Cell physiology》1997,273(4):C1194
The response ofH+-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pHi) recovery from an acid load. The rate of Na+-independentpHi recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa+-independentH+ extrusion was ATP dependent and K+ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa+-independentH+ extrusion in cultured medullarycells is mediated via H+-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H+-ATPase activity in innermedullary collecting duct cells. Upregulation ofH+-ATPase could play a protectiverole against cell death in severe intracellular acidosis. 相似文献
9.
Li G Yang Q Alexander EA Schwartz JH 《American journal of physiology. Cell physiology》2005,289(3):C665-C672
H+ transport in the collecting duct is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H+-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H+-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H+-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1AC [amino acids (aa) 1264] formed complexes with H+-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H+-ATPase (aa 235264) and another that bound SNAP23 and VAMP (aa 190234) to an equivalent degree as full-length syntaxin. However, the aa 235264 cassette alone without the SNARE N (aa 1160) does not bind but requires ligation to the SNARE N to bind H+-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H+-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H+-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H+-ATPase exocytosis. soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H++ transport 相似文献
10.
Effects of Two Plasmalemma ATPase Inhibitors on H+ Extrusion and Intracellular pH in Elodea densa Leaves 总被引:2,自引:0,他引:2
Beffagna, N. and Romani, G. 1988. Effects of two plasmalemmaATPase inhibitors on H+ extrusion and intracellular pH in Elodeadensa leaves.J. exp. Bot. 39: 10331043. Elodea leaves in the dark show very little exchange of H+ withthe medium in the external pH range between 5.0 and 6.0. Thepresence of fusicoccin and potassium in the medium markedlystimulates H+ extrusion. Fusicoccin- and K+ -induced H+ extrusionis inhibited by either erythrosin B (EB) or Na-orthovanadate,two inhibitors of H+ transporting plasma membrane ATPase. EBcompletely inhibits it from the first 30 min of treatment, whensupplied at pH 5.5 at a concentration of 30 mmol m3.Vanadate also inhibits H+ extrusion, this effect becoming evidentonly after 45 min of treatment. After this time inhibition iscomplete with 250 mmol m3 vanadate but only partial forlower concentrations. In the presence of either inhibitor the intracellular pH, measuredas cell sap pH, is significantly lowered. When the intracellularpH changes are determined on vacuole and, separately, on cytoplasmby the weak acid and base distribution method, acidificationof both compartments is found to accompany the blocking of H+extrusion by either of the inhibitors. Key words: Intracellular pH, vanadate, erythrosin B, H+pumping 相似文献
11.
ATP-dependent and PPi-dependent H+-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H+ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H+ pumping is unequivocal. H+ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg2+, the two transport systemsshowed completely different sensitivity to NO3 and K+,suggesting the presence of two types of H+-pumps in Nitellatonoplast. NO3 applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PPi-dependent transport. By contrast, NO3 appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PPi-dependent H+ transport. Replacementof K+ with the organic cation, BTP, inhibited PPi-dependenttransport but not the ATP-dependent one, indicating that PPi-dependenttransport is K+ dependent. The sensitivities of the H+ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts.
1 Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987) 相似文献
12.
Lin TY Liao BK Horng JL Yan JJ Hsiao CD Hwang PP 《American journal of physiology. Cell physiology》2008,294(5):C1250-C1260
H+-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+ activity and increased Na+ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+ uptake and acid-base regulation mechanisms in zebrafish HR cells. ionocytes; Na+/H+ exchanger; skin; gill; embryo 相似文献
13.
The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H3BO3), Na+, and Cl fluxes. Neither Na+ nor Cl appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent Km 12 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H+ gradient. Addition of 30 mM HCO3 does not affect boron extrusion by Bor1p, indicating that HCO3 does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H3BO3 causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w 相似文献
14.
The light-induced H+ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H+ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H+ efflux. Inhibitionof the H+ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H+ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH+ efflux.
1The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984) 相似文献
15.
Uozumi N 《American journal of physiology. Cell physiology》2001,281(3):C733-C739
The value of theEscherichia coli expression system has long been establishedbecause of its effectiveness in characterizing the structure andfunction of exogenously expressed proteins. When eukaryotic membraneproteins are functionally expressed in E. coli, thisorganism can serve as an alternative to eukaryotic host cells. A fewexamples have been reported of functional expression of animal andplant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologousK+ transporters exist in prokaryotic cells and ineukaryotic cells; 2) plant K+ transporters canfunctionally complement mutant K+ transporter genes inE. coli; and 3) membrane structures of plant K+ transporters can be elucidated in an E. colisystem. These experimental findings suggest the possibility ofutilizing the E. coli bacterium as an expression system forother eukaryotic membrane transport proteins. 相似文献
16.
ANDERSON W. P; WILLCOCKS D. A.; WRIGHT BARBARA J. 《Journal of experimental botany》1977,28(4):894-901
The ion contents and membrane potentials of the cells of young,hydroponically cultured seedlings of Atriplex hastata L. var.salina, Wallr. have been measured at several different NaClconcentrations. The total tissue concentrations of Na+ and Clincrease as external NaCl increases, but there is always a markedexcess of internal Na+ over Cl; this is balanced by endogenousorganic anion formation with a concomitant extrusion of H+ tothe bathing solution. Membrane potentials of the root cells remain essentially invariantwith changes in external NaCl at approx. 130 mV; thereis no evidence of a radial gradient of potential across theroot. The potential seems to contain a cyanide-sensitive electrogeniccomponent, also invariant with NaCl concentration, of about70 mV, and a diffusion component. The electrogenic componentseems likely to be a H+ efflux, probably through a H+ uniportATPase. 相似文献
17.
Matsumoto T Kanamoto T Otsuka M Omote H Moriyama Y 《American journal of physiology. Cell physiology》2008,294(4):C1074-C1078
Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different. 相似文献
18.
An active H+ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H+ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H+ influx increased as the pH of the medium was loweredto 2.9, indicating that H+ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H+ transport, related to respiration, pumps out theexcess H+ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H+efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH+ leakage through the plasma membrane. The light-induced H+ efflux and the respiration-dependent H+efflux were also compared in relation to the physiological roleof the active H+ efflux, especially with respect to the intracellularpH regulation in this alga.
1The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984) 相似文献
19.
20.
The effects of Na-orthovanadate, at concentrations only partiallyinhibiting net H+ extrusion, were determined on vacuolar andcytosolic pH by the weak base and weak acid distribution atequilibrium. Treatment with vanadate induces in Elodea densaleaves and in Arabidopsis thaliana seedlings a moderate acidificationof both cell sap and vacuole. Conversely, it induces an alkalinizationof cytosol, this effect being in apparent contrast with a conditionof reduced activity of the H+-transporting plasmalemma ATPase,which should be associated with a cytosolic acidification. InArabidopsis seedlings treated with vanadate, the increase inpH of both cytosol and external medium is associated with adecrease in cell sap buffer capacity, more evident for highervanadate concentrations, and particularly marked in the pH rangebetween 3·5 and 5·5. In these conditions, themalate content is strongly reduced, its decrease almost completelyaccounting for the decrease in cell sap buffer capacity. Anin vitro analysis of the vanadate effect on phosphoenolpyruvatecarboxylase indicates that the decrease in malate content seemssubstantially due to an inhibiting effect of vanadate on thisenzyme. These results stress that the in vivo use of vanadateas an inhibitor of the plasmalemma H+-ATPase must be taken withcaution; in particular, for studying the correlations betweenchanges in net H+ extrusion and changes in cytosolic pH andrelated processes. Key words: Vanadate, malate, cytosolic pH, Elodea densa, Arabidopsis thaliana 相似文献