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1.
The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes 总被引:3,自引:0,他引:3
The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle. 相似文献
2.
One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters. 总被引:1,自引:0,他引:1
Y Moriyama A Iwamoto H Hanada M Maeda M Futai 《The Journal of biological chemistry》1991,266(33):22141-22146
About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification. 相似文献
3.
The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out. 相似文献
4.
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 ((22)Na) 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 microM Na(+) mg(-1) membrane protein min(-1) at pH 6.5 and 25 degrees 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. 相似文献
5.
The H(+)-ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic-acid liposomes. Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2-Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent-saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated delta pH/delta psi. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability. 相似文献
6.
L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y. et al. (1986) J. Biol. Chem. 261, 4549). The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes. The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake. The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH). The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics. These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes. 相似文献
7.
Regulation of plasma membrane H<Superscript>+</Superscript>-ATPase activity by the membrane environment 总被引:1,自引:0,他引:1
Kasamo K 《Journal of plant research》2003,116(6):517-523
8.
Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation. 相似文献
9.
Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH. 总被引:4,自引:0,他引:4
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P Morsomme A de Kerchove d'Exaerde S De Meester D Thins A Goffeau M Boutry 《The EMBO journal》1996,15(20):5513-5526
In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway. 相似文献
10.
Y Moriyama K Amakatsu H Yamada M Y Park M Futai 《Archives of biochemistry and biophysics》1991,290(1):233-238
2-(4-Phenylpiperidino)cyclohexanol (AH-5183) and 2-bromo-alpha-ergocryptine, known inhibitors of the transport of acetylcholine and L-glutamate, respectively, into synaptic vesicles, inhibited the ATP-dependent uptake of dopamine in parallel with the dissipation of the electrochemical gradient of protons in chromaffin granule membrane vesicles. These compounds induced the release of accumulated dopamine from the vesicles. They also inhibited the ATP-dependent formation of the electrochemical gradient of protons in liposomes reconstituted with chromaffin H(+)-ATPase without affecting the activities for ATP hydrolysis, and ATP-dependent uptakes of dopamine, gamma-aminobutyrate, and glutamate into synaptic vesicles. These results indicated that 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine acted as uncouplers in the secretory vesicles. 相似文献
11.
M Rodriguez P Moreau M Paulik J Lawrence D J Morré D Morré 《Biochimica et biophysica acta》1992,1107(1):131-138
This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane. 相似文献
12.
D W Martin 《The Journal of biological chemistry》1990,265(34):20946-20951
Although the Ca2(+)-ATPase is the predominant protein species of the skeletal sarcoplasmic reticulum membrane, the functional significance of other minor protein species remains unresolved. The proposition has been tested that the membrane-bound 53-kDa glycoprotein (GP-53) may be required or significantly involved in regulating the coupling of ATP hydrolysis to Ca2+ transport by the Ca2(+)-ATPase. Ca2(+)-ATPases originating from preparations with and without GP-53 were reconstituted into phosphatidylcholine liposomes, and Ca2+ uptake and pumping efficiency were determined. The reconstituted Ca2+ pump from all preparations transported Ca2+ with high efficiency (Ca2+:ATP greater than 1.5). The results demonstrate that GP-53 is not required to couple ATP hydrolysis to Ca2+ transport. Additionally, the observed high coupling efficiency is inconsistent with GP-53 functioning as a substantial positive regulator of coupling. 相似文献
13.
Proteolytic activation of the plant plasma membrane H(+)-ATPase by removal of a terminal segment 总被引:13,自引:0,他引:13
Incubation of oat root plasma membrane vesicles in the presence of ATP with trypsin or chymotrypsin increased the rate of ATP hydrolysis and ATP-dependent proton pumping by the plasma membrane H(+)-ATPase. Proton pumping was stimulated more than 200%, whereas ATP hydrolytic activity was stimulated about 30%. The Km (ATP) for both proton pumping and ATP hydrolysis was lowered from about 0.3 mM to below 0.1 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trypsin-treated plasma membranes revealed a decrease in a 100-kDa band and the appearance of a 93-kDa band. Western blot analysis using antibodies against the H(+)-ATPase showed that both of these bands represented the H(+)-ATPase and suggested that a 7-kDa segment was released. Extensive treatment with carboxypeptidase A also activated the H(+)-ATPase indicating that the 7-kDa segment originated from the C terminus. 相似文献
14.
Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase. 相似文献
15.
Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle. 相似文献
16.
A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe 总被引:3,自引:0,他引:3
S Ulaszewski J C Van Herck J P Dufour J Kulpa B Nieuwenhuis A Goffeau 《The Journal of biological chemistry》1987,262(1):223-228
A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364). 相似文献
17.
Isolated H(+)-ATPase from chromaffin granules was reconstituted into liposomes and the resultant proteoliposomes were further purified by Ficoll density gradient centrifugation. Studies by electron microscopy showed that proteoliposomes had particle structures (average diameter, about 10 nm) on their outer surface. These particles could be removed from the proteoliposomes by cold treatment. Immuno-electron microscopy showed that these particles were recognized by antibodies against the hydrophilic sector of the enzyme. These results indicate that the H(+)-ATPase has a peripheral membrane structure similar to that of F1-ATPase. 相似文献
18.
Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation 总被引:1,自引:0,他引:1
E Goormaghtigh C Chadwick G A Scarborough 《The Journal of biological chemistry》1986,261(16):7466-7471
Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation. 相似文献
19.
C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane. 总被引:3,自引:0,他引:3
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The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane. 相似文献
20.
Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast. 相似文献