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1.
André L. Fonseca-de-Souza 《Experimental parasitology》2009,122(3):242-246
Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes. 相似文献
2.
Claudia Fernanda Dick André Luiz Araújo dos-Santos André L. Fonseca-de-Souza Juliana Rocha-Ferreira José Roberto Meyer-Fernandes 《Experimental parasitology》2010,124(4):386-393
In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration. 相似文献
3.
The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA3) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H+-ATPase, pyrophosphatase, H+-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H+-ATPase and H+-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H+-PPase whereas for H+-ATPase it may be GA3. Stimulation of enzyme activity by all hormones occurred at concentrations of 10–6 to 10–7
M.Abbreviations IAA
indole-3-acetic acid
- ABA
abscisic acid
- GA3
gibberellic acid
- H+-ATPase
adenosine triphosphatase
- H+-PPase
pyrophosphatase
- ATP
adenosine triphosphate
- Tris
Tris (hydroxymethyl)-aminomethane
- MES
(2[N-Morpholino]) ethane sulfonic acid
- EDTA
ethylene diamine tetraacetic acid
- Pi
inorganic phosphate 相似文献
4.
Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots 总被引:13,自引:12,他引:1
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A H+-translocating inorganic pyrophosphatase (H+-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H+-PPase catalyzed the electrogenic transport of H+ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb+ = K+ > Cs+; but it was inhibited by Na+ or Li+. Maximum rates of transport required at least 20 millimolar K+ and the Km for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K+-dependent PPase activity with an I50 of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H+-ATPase (e.g. a disulfonic stilbene or NO3−) had no effect on the PPase activity. Vanadate and azide were also ineffective. H+-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H+-PPase and the H+-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H+ pumping activity was strongly affected by Mg2+ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg2+, H+ pumping showed a KmaPP for PPi of 15 micromolar. The rate of H+ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H+-PPase. 相似文献
5.
Reversibility of H+-ATPase and
H+-Pyrophosphatase in Tonoplast Vesicles from Maize
Coleoptiles and Seeds
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Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to 32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation. 相似文献
6.
Ching-Hung Lee Yen-Wei Chen Yun-Tzu Huang Yih-Jiuan Pan Chien-Hsien Lee Shih-Ming Lin Lin-Kun Huang Yueh-Yu Lo Yu-Fen Huang Yu-Di Hsu Shih-Chung Yen Jenn-Kang Hwang Rong-Long Pan 《The Journal of membrane biology》2013,246(12):959-966
H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH+-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H+-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H+-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH+-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K+-mediated stimulation of H+-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase. 相似文献
7.
A unique variant strain of Chara corallina, which contains little inorganic phosphate in the vacuole ([Pi]v) was isolated. The level of cytoplasmic inorganic phosphate ([Pi]c) in these cells was the same as that in normal cells. Using these unique cells, we studied the change in [Pi]c and the effect of Pi on the activities of electrogenic H+-pumps associated with the plasma membrane and tonoplast. Upon illumination, the plasma membrane of C. corallina became hyperpolarized by 15 mV, the pH of the vacuolar sap decreased by 0.5 unit, and [Pi]c decreased by 30% with a similar time course. The activities of the electrogenic H +-pump in the plasma membrane and the ATP and PPi-dependent H+-transport in the tonoplast were noncompetitively inhibited by Pi with Ki values of, in the order given, 21.3 mM, 22.1 mM and 37.7 mM. From the kinetics study we calculated that the electrogenic H+-pump in the plasma membrane and the ATP and PPi-dependent H+ transport in the tonoplast were activated by, again in this order, 13%, 13% and 9%, in accordance with the decrease in [Pi]c. We propose that the change in [Pi]c is one of the regulators of photosynthesis-mediated activation of the H+-pumps in the plasma membrane and the tonoplast in C. corallina upon illumination. 相似文献
8.
Marco Antonio Lacerda-Abreu Thais Russo-Abrahão Daniela Cosentino-Gomes Michelle Tanny Cunha Nascimento Luiz Fernando Carvalho-Kelly Tainá Gomes Mariana Figueiredo Rodrigues Sandra König Franklin David Rumjanek Robson Q. Monteiro José Roberto Meyer-Fernandes 《生物化学与生物物理学报:疾病的分子基础》2019,1865(9):2180-2188
Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ± 0.1674 mM Pi and Vmax = 198.6 ± 10.23 Pi × h?1 × mg protein?1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes. 相似文献
9.
Ohnishi M Mimura T Tsujimura T Mitsuhashi N Washitani-Nemoto S Maeshima M Martinoia E 《Planta》2007,225(3):711-718
Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to
high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H+ pumping, we examined the dependency of H+ pumping on plant Pi status. Both H+ pumping and the activities of the vacuolar H+-pumps, the V-type H+-ATPase and the H+-PPase were enhanced under low Pi status. Despite this increase in H+ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for
the activation of Pi uptake into the vacuole under low Pi status are discussed.
Miwa Ohnishi and Tetsuro Mimura contributed equally to this work. 相似文献
10.
Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors 总被引:3,自引:3,他引:0
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The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H+-PPase) from oat (Avena sativa L.) roots to changes in Mg2+ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H+-PPase is Mg2PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg2+ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H+-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H+-PPase. It is demonstrated that models incorporating Mg2PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H+-PPase. Calculations of the likely concentrations of Mg2PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H+-PPase would be about 70% saturated in vivo. 相似文献
11.
Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis 总被引:3,自引:1,他引:2
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Rea PA Britten CJ Jennings IR Calvert CM Skiera LA Leigh RA Sanders D 《Plant physiology》1992,100(4):1706-1715
The H+-translocating inorganic pyrophosphatase (H+-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2+ and a number of other divalent cations (Co2+, Mn2+, Zn2+). By contrast, the H+-translocating ATPase (H+-ATPase) located on the same membrane is insensitive to Ca2+. Here we examine the mechanism and feasibility of regulation of the vacuolar H+-PPase by cytosolic free Ca2+ under the conditions thought to prevail in vivo with respect to Mg2+, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca2+ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg2PPi) at one site, inhibitory binding of Mg2PPi to a lower-affinity second site, binding of activator (Mg2+) at a third site, and direct binding of Ca2+ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca2+ or CaPPi are explicable only if Ca2+, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca2+ on the substrate concentration dependence of H+-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H+-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca2+ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells. 相似文献
12.
The simple proton-translocating inorganic pyrophosphatase (H+-PPase) found in plants and protists is an evolutionally conserved, essential enzyme that catalyzes the hydrolysis of pyrophosphate (PPi). Little is known about the functional contribution of H+-PPase to the cellular response to abiotic stresses, except its high salinity and drought stress. To investigate the role of H+-PPase during response to cellular stress, we isolated the cDNA of Arabidopsis thaliana H+-PPase (AVP1) and Oryza sativa H+-PPase (OVP1) and constructed transgenic Saccharomyces cerevisiae and Escherichia coli lines that express AVP1 and OVP1. In S. cerevisiae, the expression of a chimeric derivative of the AVP1 and OVP1 alleviated the phenotype associated with ipp2-deficient cells in the presence of high salinity (NaCl) and metal stressors (Cd, Mn, and Zn). In E. coli, AVP1 and OVP1 overexpression conferred enhanced tolerance to abiotic stresses, including heat shock and H2O2, as well as NaCl, Cd, Mn, Zn, Ca, and Al. Interestingly, AVP1 and OVP1 overexpression resulted in hypersensitivity to menadione and cobalt. These results demonstrate the cellular capacity of AVP1- and OVP1-expressing transgenic yeast and E. coli in response to physiological, abiotic stresses. Moreover, our results suggest new ways of engineering stress-tolerant plants that are capable of responding to climate change. Here, we provide an outline of an experimental system to examine the alternative roles of plant H+-PPase. 相似文献
13.
Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue
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
14.
Yun-Tzu Huang Tseng-Huang Liu Shih-Ming Lin Yen-Wei Chen Yih-Jiuan Pan Ching-Hung Lee Yuh-Ju Sun Fan-Gang Tseng Rong-Long Pan 《The Journal of biological chemistry》2013,288(27):19312-19320
Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H+-PPase consists of 14–16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H+-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H+-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H+-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H+-PPase upon substrate binding. 相似文献
15.
H+-translocating inorganic pyrophosphatases (H+-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H+-PPase could be an early-originated protein that participated in many important biochemical and physiological processes. In this study we cloned 14 novel sequences from 7 eremophytes: Sophora alopecuroid (Sa), Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi), Suaeda salsa (Ss), Suaeda rigida (Sr), Halostachys caspica (Hc), and Karelinia caspia (Kc). These novel sequences included 6 ORFs and 8 fragments, and they were identified as H+-PPases based on the typical conserved domains. Besides the identified domains, sequence alignment showed that there still were two novel conserved motifs. A phylogenetic tree was constructed, including the 14 novel H+-PPase amino acid sequences and the other 34 identified H+-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these 48 H+-PPases were classified into two groups: type I and type II H+-PPase. The novel 14 eremophyte H+-PPases were classified into the type I H+-PPase. The 3D structures of these H+-PPase proteins were predicted, which suggested that all type I H+-PPases from higher plants and algae were homodimers, while other type I H+-PPases from bacteria and protozoans and all type II H+-PPases were monomers. The 3D structures of these novel H+-PPases were homodimers except for SaVP3, which was a monomer. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H+-PPase family. 相似文献
16.
Julio Paez-Valencia Araceli Patron-Soberano Alejandra Rodriguez-LevizJonathan Sanchez-Lares Concepcion Sanchez-GomezPedro Valencia-Mayoral Guadalupe Diaz-RosasRoberto Gaxiola 《Plant science》2011,181(1):23-30
Previous literature has shown the presence of a plasma membrane (PM) localized type I H+-PPase in sieve elements of Ricinus communis. Unfortunately, the physiological relevance of these findings remains obscure due to the lack of genetic and molecular reagents to study R. communis. The availability of H+-PPase gain and loss-of-function mutants in Arabidopsis thaliana makes this plant an attractive genetic model to address the question, but data on the PM localization of this H+-PPase in A. thaliana are limited to two proteomic approaches. Here we present the first report on the localization of the type I H+-PPase AVP1 in sieve element-companion cell complexes (SE-CCc) from A. thaliana. Double epifluorescence and immunogold labeling experiments are consistent with the co-localization of AVP1 and PIP1 (a bona fide PM maker) in PM of SE-CCc from A. thaliana. 相似文献
17.
《Journal of molecular biology》2019,431(8):1619-1632
Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH+-PPase), the VrH+-PPase–2Pi complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH+-PPases complexes (Pi-, 2Pi- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2Pi-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH+-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of VrH+-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer. 相似文献
18.
Hideaki Matsumoto Yasushi Senoo Minobu Kasai Masayoshi Maeshima 《Journal of plant research》1996,109(1):99-105
Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis
was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed
Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations
of Ca2+ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl2 completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca2+ was due to the reduction of Al uptake.
H+-ATPase and H+-PPase activity as well as ATP and PPidependent H+ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment
with 50 μM AlCl3. The rate of increase of the amount of H+-ATPase and H+-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit
of the vacuolar H+-ATPase and H+-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of
barley root against Al stress. 相似文献
19.
Vacuoles isolated from storage roots of red beet (Beta vulgaris L.) posess a Mg2+-dependent, alkaline pyrophosphatase (PPase) activity which is further stimulated by salts of monovalent cations. The requirement for Mg2+ is specific. Mn2+ and Zn2+ permitted only 20% and 12%, respectively, of the PPase activity obtained in the presence of Mg2+ while Ca2+, Co2+ and Cu2+ were ineffective. Stimulation of Mg2+-PPase activity by salts of certain monovalent cations was due to the cation and the order of effectiveness of the cations tested was K+=Rb+=NH
4
+
>Cs+. Salts of Li+ and Na+ inhibited Mg2+-PPase activity by 44% and 24%, respectively. KCl-stimulation of Mg2+-PPase activity was maximal with 60–100 mM KCl. There was a sigmoidal relationship between PPase activity and Mg2+ concentrations which resulted in markedly non-linear Lineweaver-Burk plots. At pH 8.0, the optimal [Mg2+]:[PPi] ratio for both Mg2+-PPase and (Mg2++KCl)-PPase activities was approximately 1:1, which probably indicates MgP2O7
2- is the true substrate.Abbreviations BSA
bovine serum albumen
- EDTA
ethylenediamine tetra-acetic acid, disodium salt
- MES
2-(N-morpholino)ethanesulphonic acid
- Mg
T
2+
total magnesium
- Pi
inorganic phosphate
- PPase
inorganic pyrophosphatase
- PPi
inorganic pyrophosphate
- TCA
trichloroacetic acid
- Tris
tris(hydroxymethyl)methylamine 相似文献
20.
Yen-Wei Chen Ching-Hung Lee Yun-Tzu Huang Yih-Jiuan Pan Shih-Ming Lin Yueh-Yu Lo Chien-Hsien Lee Lin-Kun Huang Yu-Fen Huang Yu-Di Hsu Rong-Long Pan 《Journal of bioenergetics and biomembranes》2014,46(2):127-134
Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis. 相似文献