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1.
The monoclonal antibody to the β-subunit of H+/K+-ATPase (mAbHKβ) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca2+-ATPase. We partially purified a mAbHKβ-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca2+-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca2+-ATPase. Synthesis of functional SR Ca2+-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca2+-ATPase synthesis.  相似文献   

2.
Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na+ + K+)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ1 heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na+ + K+)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na+ + K+)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na+ + K+)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.  相似文献   

3.
The first evidence of autoproteolytic activity of the ~50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their ~100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183–1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249–Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221–231; Ahmed et al. (2003). Biochemistry 42:12539–12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (–P1–P1′ –) in one serotype (except Asp–Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249–Tyr250, Gln258–Ser259 and Ile243–Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT’s structure–function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418–Thr419 or Phe422–Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT’s entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs—the 9-residuelong motifs present on the NT’s natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD+/NADH associated with the NTs type A, B and E (2–3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase—a representative NAD+/NADH binding protein.  相似文献   

4.
The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H+-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K+ channel (unit conductance 5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1in (for Coleoptile K + Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca2+ level. Whereas at nanomolar Ca2+ concentrations K+ currents increased with a t1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K+ concentration the reversal potential of the K+ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na+, Rb+, Cs+ and a permeability ratio PNH 4 + /PK+ around 0.25 underlines the high selectivity of CKC1in for K+In contrast to Cs+, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb+, often used as a tracer for K+, does not permeate this type of K+ channelThe lack of Rb+ permeability is unique with respect to other K+ transporters. Therefore, future molecular analysis of CKC1in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K+ channels in general.Abbreviations CKC1in Coleoptile K + Channel inward rectifier - U membrane voltage - Iss steady-state currents - Itail tail currents Experiments were conducted in the laboratory of F.G. during the stay of RHas a guest professor sponsored by Special Project RAISA, subproject N2.1, paper N2155.  相似文献   

5.
Populus euphratica is a salt-tolerant tree species growing in semi-arid saline areas. A Na+/H+ antiporter gene was successfully isolated from this species through RACE cloning, and named PeSOS1. The isolated cDNA was 3665 bp long and contained a 3438 bp open reading frame that was predicted to encode a 127-kDa protein with 12 hypothetical transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The amino acid sequence of this PeSOS1 gene showed 64% identity with the previously isolated SOS1 gene from the glycophyte Arabidopsis thaliana. The level of protein expressed by PeSOS1 in the leaves of P. euphratica was significantly up-regulated in the presence of high (200 mM) concentrations of NaCl, while the mRNA level in the leaves remained relatively constant. Immunoanalysis suggested that the protein encoded by PeSOS1 is localized in the plasma membrane. Expression of PeSOS1 partially suppressed the salt sensitive phenotypes of the EP432 bacterial strain, which lacks the activity of the two Na+/H+ antiporters EcNhaA and EcNhaB. These results suggest that PeSOS1 may play an essential role in the salt tolerance of P. euphratica and may be useful for improving salt tolerance in other tree species. Yuxia Wu and Nan Ding contributed equally to this work.  相似文献   

6.
Proton transport in the terminal part of the respiratory chain in the extremely alkaliphilic halotolerant bacterial strain Thioalkalivibrio versutus was studied under near-optimum growth conditions (pH 9.0-9.5). Under these conditions, bacterial cells generated electric potential with the negative charge being inside the cells. When only the terminal part of the respiratory chain functioned, it was found that: 1) unlike other bacteria known, this bacterium did not acidify the medium in the presence of K+ and valinomycin; 2) in the presence of an uncoupler, CCCP, but in the absence of valinomycin, reversible alkalinization of the medium occurred as a result of proton influx into the cells. Cyanide prevented this alkalinization. The difference spectra indicate that cell membranes contained cytochromes c and (b + o), some of which reacted with CO. The respiratory activity of membranes in the terminal part of the respiratory chain was optimal at pH 9.5 and specifically depended on sodium ions (C 1/2 = 10 mM). The data suggest the presence of a Na+-pump in the terminal part of the respiratory chain of the studied strain which can pump Na+ out of the cells.  相似文献   

7.
We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.  相似文献   

8.
Li Z  Zhang X 《Planta》2004,219(6):948-954
The vacuolar H+-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V1 and Vo with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V1 and Vo domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.Abbreviations MSA Multivariate statistical analysis - 2D, 3D Two-, three-dimensional - V-ATPase Vacuolar H+-ATPase  相似文献   

9.
Achim Hager  Christa Lanz 《Planta》1989,180(1):116-122
Functional properties and the localization of essential SH-groups of the tonoplast H+-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H+-translocation activity of the tonoplast, the H+-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl2 and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H+ pump—avoided the inhibition of the H+-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H+-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H+-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.  相似文献   

10.
Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pK a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes V max but not K m values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.  相似文献   

11.
J. Wang  K. Zuo  W. Wu  J. Song  X. Sun  J. Lin  X. Li  K. Tang 《Biologia Plantarum》2004,48(4):509-515
Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na+/H+ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T1 progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T1 lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T1 progenies could express BnNHX1 and had salt tolerance at levels comparable to their T0 parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.  相似文献   

12.
Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na+-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na+, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na+ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K+-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K+. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K+ and Rb+ transporter. CnSOS1B mediated a transient, extremely rapid K+ or Rb+ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K+ and Rb+ uptake characteristics in bacteria.  相似文献   

13.
Summary The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K+-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (–47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Ca i ) from 200 to 2 nm or increasing the external [Ca2+] (Ca o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Ca o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Ca i -, and Ca o -dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectiying K+ channels are part of a regulatory mechanism for Ca i . Changes in Ca o and (associated) changes in Ca i regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.  相似文献   

14.
The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (15NH4)2SO4 ranging from 10 to 200 M NH 4 + with and without 200 M K(86Rb)Cl or to K(86Rb)Cl ranging from 10 to 200 M K+ with and without 200 M NH 4 + as (15NH4)2SO4. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H+ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V max and increases in Km of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned  相似文献   

15.
Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617–9624). In the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP+ at rates of 333 and 110 mol (mg bacteriochlorophyll a)–1 h–1, respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c 551 and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c 551 inhibit NADP+ reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.Abbreviations FMO protein Fenna-Matthews-Olson protein - TMBZ 3,3,5,5-tetramethylbenzidine  相似文献   

16.
The toxic effect of Pb2+ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P m) and the growth rate constant (k) were determined, which showed that values of P m and k were linked to the concentration (C) of Pb2+. The addition of Pb2+ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb2+, and Pb2+ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb2+, indicating that the toxic effect of Pb2+ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb2+.  相似文献   

17.
The role of H2O2 in abscisic acid (ABA)-induced NH4+ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH4+ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH4+ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H2O2, was observed to be effective in inhibiting ABA-induced accumulation of NH4+ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H2O2 contents in rice leaves, inhibited ABA-induced increase in the content of NH4+. Similarly, the changes of enzymes responsible for NH4+ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H2O2 was found to increase NH4+ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H2O2 is involved in ABA-induced NH4+ accumulation in rice leaves.  相似文献   

18.
Fusicoccin (FC) has long been known to promote K+ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H+ pumping stimulated in FC, could help drive K+ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K+ (86Rb+) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K+ movement across the membrane could be modified in FC. We have explored a role for K+ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (86Rb+) flux and in electrical current under voltage clamp was followed, using the K+ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K+-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K+ channel current contributed appreciably to charge balance maintainingV m, and adding 10 mM TEA to block the current depolarized (positive-going)V m; TEA also reduced86Rb+ efflux by 68–80%. Following treatments with 10 M FC, both K+ channel current and86Rb+ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced86Rb+ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K+ (Rb+), despite membrane potentials which were 30–60 mVpositive of the K+ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K+ channels. They offer evidence of an energy-coupled mechanism for K+ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K+ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E K equilibrium potential for K+ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G m membrane (slope) conductance atV m - I-V current-voltage (relationship) - apparent rate constant for exchange - K i + , K 0 + intracellular, extracellular K+ (concentration) - TEA tetraethylammonium chloride - V m free-running membrane potential (difference)  相似文献   

19.
Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH i =6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pH o =6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pH i =6.4) markedly increased when pH o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pH0 was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pH i was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Na i + =Na 0 + =15mm,Em=+6.7 mV), an outward H+ gradient (pH i =6.4, pH0=8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.  相似文献   

20.
pH i recovery in acid-loaded Ehrlich ascites tumor cells and pH i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO 3 -free media and at 25 mm HCO 3 , the measured pH i (7.26 and 7.82, respectively) was significantly more alkaline than the pH i . value calculated assuming the transmembrane HCO 3 gradient to be equal to the Cl gradient. Thus, pH i in these cells is not determined by the Cl gradient and by Cl/HCO 3 exchange.pH i recovery following acid loading by propionate exposure, NH 4 + withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3 free media, and in the presence of HCO 3 (25 mm) by DIDS-sensitive, Na+-dependent Cl/HCO 3 exchange. A significant residual pH i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pH i regulation. pH i maintenance at steady-state involves both Na+/H+ exchange and Na+-dependent Cl/HCO 3 exchange.Acute removal of external Cl induces a DIDS-sensitive, Na+-dependent alkalinization, taken to represent HCO 3 influx in exchange for cellular Cl. Measurements of 36Cl efflux into Cl-free gluconate media with and without Na+ and/or HCO 3 (10 mm) directly demonstrate a DIDS-sensitive, Na+ dependent Cl/HCO 3 exchange operating at slightly acidic pH i (pHo 6.8), and a DIDS-sensitive, Na+-independent Cl/HCO 3 exchange operating at alkaline pH i (pH o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).  相似文献   

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