共查询到20条相似文献,搜索用时 31 毫秒
1.
The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H +-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H +-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H +-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H +-ATPase, therefore increasing the catalytic activity of the phosphatase domain. 相似文献
2.
The effect of fusicoccin (FC) on the activity of the PM H +-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H +-ATPase activity by up to 100 %; the effect was essentially on V max with only a slight decrease of the apparent K M of the enzyme for ATP. FC-induced stimulation of the PM H +-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H +-ATPase is very rapid. Both FC-induced stimulation of the PM H +-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H +-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H +-ATPase. 相似文献
3.
The plasma membrane (PM) H + ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice ( Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H + ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H + ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value ( K m ) of PM H + ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H + ATPase in P deficient roots was related to a slightly increase of PM H + ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H +-ATPase and H + pump, which contributed to the rhizosphere acidification in rice roots. 相似文献
4.
Membranes of corn ( Zea mays, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient centrifugation and the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase was selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelles. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCl, Mg 2+-ATPase of the PM-rich fraction from a step gradient was 6.0 to 6.5, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To reduce PM contamination, Golgi-rich fractions obtained from step or rate-zonal gradients were recentrifuged isopycnically on linear sucrose gradients. The distribution of KCl, Mg 2+-ATPase activity was measured at pH 6.5 and 7.5. The pH 6.5 ATPase was coincident with UDPG-sterol glucosyl transferase, a PM marker, while the pH 7.5 ATPase overlapped with latent UDPase, a Golgi marker. These results provide strong evidence for a KCl, Mg 2+-ATPase, active at pH 7.5, associated with the Golgi membranes of corn coleoptiles. 相似文献
5.
The stimulation of the plasma membrane (PM) H +-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 m M boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H + transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H + transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H +-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 m M boron such hyperpolarizations, perhaps caused by an enhanced activity of the H +-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V m may be reduced by additional stimulation of a K + inward current of opposite direction to the H +-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H +-ATPase. A boron-increased PM H +-ATPase activity in turn may stimulate germination and growth of pollen tubes. 相似文献
6.
Based on hydropathy analysis, the P-type cation translocating ATPases are believed to have similar topological arrangements in the membrane, but little independent evidence exists for their precise pattern of transmembrane folding. As a first step toward defining the topology of the Neurospora plasma membrane H+-ATPase, we have mapped the orientation of the amino and carboxyl termini. In three different types of experiments, both termini of the H+-ATPase were shown to be exposed at the cytoplasmic surface of the plasma membrane: 1) antibodies specific for the amino and carboxyl termini bound to permeabilized but not intact cells; 2) inside-out plasma membrane vesicles were approximately 100-fold more effective than intact cells in competing for antibody binding; and 3) trypsin, which is known to proteolyze three sites at the amino terminus and one site at the carboxyl terminus of the purified Neurospora H+-ATPase (Mandala, S. M., and Slayman, C. W. (1988) J. Biol. Chem. 263, 15122-15128), was found in the present study to cleave the same sites in inside-out plasma membrane vesicles but not in intact cells. These results indicate that the ATPase polypeptide traverses the membrane an even number of times, in support of a previously published topological model (Hager, K. M., Mandala, S. M., Davenport, J. W., Speicher, D. W., Benz, E. J., Jr., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697). 相似文献
7.
Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H +-ATPase in plasma membrane (PM) isolated from radish ( Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H +-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H +-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H +-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H +-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H +-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H +-ATPase. Analysis of the activation state of PM H +-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme. 相似文献
8.
We have isolated the plasma membrane H +−ATPase in a phosphorylated form from spinach ( Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H +-ATPase, thus activating H + pumping. We have identified threonine-948, the second residue from the C-terminal end of the H +-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H +-ATPase. 相似文献
9.
The determination of the crystal structure of the Ca 2+-ATPase of sarcoplasmic reticulum (SR) in its Ca 2+-bound [Nature 405 (2000) 647] and Ca 2+-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca 2+-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM α-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 Å, rather than the normal 30 Å. The experimentally determined TM α-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM α-helices in the Ca 2+-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM α-helices, with changes in TM α-helical packing being important in the process of transfer of Ca 2+ ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM α-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca 2+-ATPase. 相似文献
10.
The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn ( Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat ( Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase. Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K+. K+-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate. 相似文献
11.
In situ plasma membrane H +-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H +-ATPase activity as compared to the yeast form. At the time of evagination H +-efflux exhibited an increase. K + depletion resulted in attenuated ATPase activity and glucose induced H +-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K + acts as a modulator.Abbreviations PM
Plasma membrane
- pHi
intracellular pH
- Pi
inorganic phosphorus
- TET
Toluene: Ethanol: Triton X-100 相似文献
12.
Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H +-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H +-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump. 相似文献
13.
The influence of freezing treatment on plasma membrane (PM) H +-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (−4 °C) for 5 d resulted in significant
increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was
less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine
was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but
increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that
PM H +-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana. 相似文献
14.
Two distinct membrane fractions containing H +-ATPase activity were prepared from red beet. One fraction contained a H +-ATPase activity that was inhibited by NO 3− while the other contained a H +-ATPase inhibited by vanadate. We have previously proposed that these H +-ATPases are associated with tonoplast (NO 3−-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP 2−, and Mg 2+ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP ( i.e. ATP concentrations in excess of Mg 2+) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP. Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP2− in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner. Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP2− complex. The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H+-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg2+ could modulate ATPase activity at both the tonoplast and plasma membrane. 相似文献
16.
We have estimated the amount of inside-out plasma membrane (PM) vesicles in microsomal fractions from wheat ( Triticum aestivum L. cv. Drabant) and maize ( Zea mays L.) roots; non-latent activities of the PM markers vanadate-inhibited K +, Mg 2+-ATPase (ΔVO 4-ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside-out PM vesicles, latent activities as markers for right-side-out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside-out PM vesicles from right-side-out ones was achieved by counter-current-distribution (CCD) in an aqueous polymer two-phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA-stained vesicles. Thus, most of the PM vesicles had a right-side-out orientation. ΔVO 4-ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO 4-ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO 4-ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right-side-out PM vesicles. Taken together, these results indicate that only very few inside-out PM vesicles are present in the microsomal fraction, and that ΔVO 4-ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well-defined preparations of scaled, inside-out PM vesicles in solute uptake studies. The distribution of Ca 2+-inhibited ATPase, on the other hand, agreed well with those of GS II and STA-stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca 2+ inhibition may be a more specific property of the PM H +-ATPase than vanadate inhibition. 相似文献
17.
Spartina patens, an intertidal C4 grass, grows in the
upper salt marsh and tolerates coastal seawater salinity. The regulation of
ion movement across the plasma membrane (PM) for plant salt tolerance is
thought to be achieved by an electrochemical gradient generated by plasma
membrane H +-ATPase. In this study, the change of PM
H +-ATPase in response to NaCl was characterized for
S. patens callus. Callus was cultured for 10 weeks
under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma
membrane was isolated from a Dextran/PEG aqueous polymer two-phase system
and the purity was demonstrated with membrane enzyme markers. There was a
significant increase (up to 2-3 fold) of PM
H +-ATPase activity when callus was grown on media
containing NaCl. The incremental activation of PM
H +-ATPase activity would enable the cell to tolerate
higher cytoplasmic NaCl concentrations. PM H +-ATPase
appeared to have a higher Vmax and a lower substrate
concentration ( Km to reach Vmax.
When growth medium salinity increased from 0 mM to 170 and 340 mM, the
Vmax of H +-ATPase increased
from 0.64 to 1.00 and 1.73, respectively, while the Km
decreased from 3.58 to 2.07 and 2.44 mM, respectively. In
vitro NaCl inhibition kinetic data revealed a pattern of
non-competitive inhibition by NaCl on PM H +-ATPase.
The response of PM H +-ATPase in S.
patens callus suggests that this species has evolved mechanisms
that can regulate this important enzyme when cells are exposed to
NaCl. 相似文献
18.
The plasma membrane (PM) vesicles from Populus euphratica ( P. euphratica) callus were isolated to investigate the properties of the PM H +-ATPase. An enrichment of sealed and oriented right-side-out PM vesicles was demonstrated by measurement of the purity and
orientation of membrane vesicles in the upper phase fraction. Analysis of pH optimum, temperature effects and kinetic properties
showed that the properties of the PM H +-ATPase from woody plant P. euphratica callus were consistent with those from herbaceous species. Application of various thiol reagents to the reaction revealed
that reduced thiol groups were essential to maintain the PM H +-ATPase activity. In addition, there was increased H +-ATPase activity in the PM vesicles when callus was exposed to NaCl. Western blotting analysis demonstrated an enhancement
of H +-ATPase content in NaCl-treated P. euphratica callus compared with the control. 相似文献
19.
Purified plasma membranes from several transplantable human tumors exhibit very high Mg 2+-dependent ATPase activities. Three types of Mg 2+-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na +, K +-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg 2+-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na + (or K +) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg 2+-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed. 相似文献
20.
The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate-sensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive H + pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca 2+: sensitivity to vanadate (IC 50 ca. 1 μM), Mg 2+-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H +-ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP. 相似文献
|