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1.
Calcium transport into tomato ( Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca 2+ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca 2+. A low affinity Ca 2+ uptake system ( Km > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H +/Ca 2+ antiport. A high affinity Ca 2+ uptake system ( Km = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca 2+ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca 2+/H + antiport activity could only by partially ascribed to an effect of low temperature on H +-ATPase activity, ATP-dependent H + transport, passive H + fluxes, or passive Ca 2+ fluxes. These results suggest that low temperature directly affects Ca 2+/H + exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca 2+/H + exchange protein or by an indirect effect of temperature on lipid interactions with the Ca 2+/H + exchange protein. 相似文献
2.
Two types of ATP-dependent calcium (Ca 2+) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca 2+ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO 3−. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg 2+. Like the tonoplast H+-ATPase activity, vanadate-insensitive Ca2+ accumulation was stimulated by 20 millimolar Cl− and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca2+ transport system had an apparent Km for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca2+ transport was abolished by compounds that eliminated a pH gradient and Ca2+ dissipated a pH gradient (acid inside) generated by the tonoplast-type H+-ATPase. These results provide compelling evidence that a pH gradient generated by the H+-ATPase drives Ca2+ accumulation into right-side-out tonoplast vesicles via a Ca2+/H+ antiport. This transport system was saturable with respect to Ca2+ (Km apparent = 14 micromolar). The Ca2+/H+ antiport operated independently of the H+-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca2+ accumulation. Ca2+ transport by this system may be one major way in which vacuoles function in Ca2+ homeostasis in the cytoplasm of plant cells. 相似文献
3.
Calcium uptake by microsomal membranes from the cellular slime mould Dictyostelium discoideum was measured using Calcium Green-2 as a fluorescent probe of external free Ca 2+ concentration. High-affinity Ca 2+ uptake was found to be completely inhibited by low concentrations of vanadate, but not by thapsigargin, suggesting that the activity is mediated by a Ca 2+-ATPase distinct from sarco(endo)plasmic reticulum type of higher animal cells. On sucrose density gradients, Ca 2+ uptake distributes with vacuolar proton pump activity and part of the observed Ca 2+ uptake is dependent on the pH gradient generated by the vacuolar-type H +-ATPase, indicating that the Ca 2+ pump is located on both acidic and non-acidic vesicles, possibly derived from the H +-ATPase-rich contractile vacuole complex. 相似文献
4.
Microsomal vesicles from 24-hour-old radish ( Raphanus sativus L.) seedlings accumulate Ca 2+ upon addition of MgATP. MgATP-dependent Ca 2+ uptake co-migrates with the plasma membrane H +-ATPase on a sucrose gradient. Ca 2+ uptake is insensitive to oligomycin, inhibited by vanadate (IC 50 40 micromolar) and erythrosin B (IC 50 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca 2+ uptake is insensitive to protonophores. These results indicate that Ca 2+ transport in these microsomal vesicles is catalyzed by a Mg 2+-dependent ATPase localized on the plasma membrane. Ca 2+ strongly reduces ΔpH generation by the plasma membrane H +-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca 2+ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca 2+ uptake into plasma membrane vesicles. The Ca 2+-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca 2+-induced decrease of ΔpH generation by the plasma membrane H +-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H +-ATPase, is accelerated by MgATP-dependent Ca 2+ uptake, indicating that the decrease of ΔpH generation induced by Ca 2+ reflects the efflux of H + coupled to Ca 2+ uptake into plasma membrane vesicles. It is therefore proposed that Ca 2+ transport at the plasma membrane is mediated by a Mg 2+-dependent ATPase which catalyzes a nH +/Ca 2+ exchange. 相似文献
5.
Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca 2+ and H + transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize ( Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca 2+ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H + gradient was created on the tonoplast vesicles by means of H +-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca 2+ and H + across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed. 相似文献
6.
Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U 3+U 3′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven 45Ca 2+ transport [? 15 pkat (mg protein) −1]. Ca 2+ uptake into the vesicles had a pH optimum of 7.2 and apparent K m values for Ca 2+ of 4.4 μ M and for Mg-ATP of 300 μ M. Ca 2+ uptake, K +, Mg 2+-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm −3) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca 2+ transport into the vesicles, excluding a Ca 2+/H + exchange driven by a proton gradient. ATP-dependent Ca 2+ uptake was inhibited by erythrosin B (I 50= 0.1 μ M), ruthenium red (I 50= 30 μ M), La 3+ (I 50= 10 μ M) and vanadate (I 50= 500 μ M), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I 50= 70 μ M) and W-7 (I 50= 100 μ M) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca 2+ efflux from the vesicles rather than inhibit Ca 2+ uptake. Our results demonstrate the presence of a Ca 2+-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca 2+ out of the cell. Its high affinity for Ca 2+ makes it a likely component involved in adjusting low cytoplasmic Ca 2+ levels. No indications for a secondary active Ca 2+/H + transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca 2+-ATPase from the H +-translocating K +. Mg 2+-ATPase in C. communis plasma membranes. 相似文献
7.
The primary or secondary energized transport of Ca 2+, Mg 2+ and H + into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca 2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca 2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca 2+-pumping and tonoplast H +-pyrophosphatase (PP iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca 2+-pumping activity could be detected. The ATP-dependent Ca 2+ uptake into tonoplast vesicles (using Ca 2+ concentrations from 0.8–1 μM) was completely inhibited by the Ca 2+ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H + accumulation had no effect. Vanadate (IC 50 = 43 μM) and diethylstilbesterol (IC 50 = 5.2 μM) were potent inhibitors of this type of Ca 2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity ( K m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca 2+ transport mechanism is the only functioning Ca 2+ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca 2+ concentration. In contrast, the secondary energized Ca 2+-transport mechanism of the tonoplast, the low-affinity Ca 2+/H +-antiporter, which was reported to allow the uptake of Ca 2+ in exchange for H +, functions chiefly as an Mg 2+ transporter under physiological conditions because cytosolic Mg 2+ is several orders of magnitude higher than the Ca 2+ concentration. This conclusion was deduced from experiments showing that Mg 2+ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H + from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H +-ATPase and H +-PP iase was found to be influenced by Ca 2+ differently from and independently of the Mg 2+ concentration. Calcium was a strong inhibitor for the H +-PP iase (IC 50 = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H +-ATPase (IC 50 = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca 2+-and Mg 2+-regulated H +-PP iase, (ii) the newly described high-affinity Ca 2+-AT-Pase, (iii) the low-affinity Mg 2+(Ca 2+)/H +-antiporter and (iv) the H 2+-ATPase. 相似文献
8.
An anion-sensitive H +-translocating ATPase was identified in membrane vesicles isolated from mature green tomato ( Lycopersicon esculentum) fruit. The H +-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO 3−, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H +-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The Km for the H +-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H +-ATPase are very similar to those described for a number of plant cell tonoplast H +-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation. 相似文献
9.
Two active calcium (Ca 2+) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells ( Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca 2+ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl −-stimulated, NO 3−-inhibited ATPase activity on sucrose density gradients. Ca 2+ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide- m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The K m for MgATP and Ca 2+ were 0.1 m m and 21 micromolar, respectively. The predominant Ca 2+ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca 2+ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I 50 = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca 2+ dependent kinetics were complex with an apparent K m ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca 2+/H + antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca 2+ transport systems are proposed to restore and maintain cytoplasmic Ca 2+ homeostasis under changing cellular and environmental conditions. 相似文献
10.
Sealed microsomal vesicles were prepared from corn ( Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000 g microsomal fraction onto a 10% dextran T-70 cushion. The Mg 2+-ATPase activity of the sealed vesicles was stimulated by Cl − and NH 4+ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO 3−, was insensitive to K +, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by Cl−, inhibited by NO3−, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg2+ ≥ Mn2+ > Co2+) and were inhibited by high concentrations of Ca2+. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F1F0-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. 相似文献
11.
A large number of plant Ca 2+/H + exchangers have been identified in endomembranes, but far fewer have been studied for Ca 2+/H + exchange in plasma membrane so far. To investigate the Ca 2+/H + exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca 2+/H + exchange in plasma membrane vesicles was measured by Ca 2+-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca 2+/H + exchange was optimal at pH 7.0, and displayed transport specificity for Ca 2+ with saturation kinetics at K m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca 2+-dependent transport of H + out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca 2+/H + exchange increased comparing to control without valinomycin effect, suggesting that the Ca 2+/H + exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H +:Ca 2+. Eosin Y, a Ca 2+-ATPase inhibitor, drastically inhibited Ca 2+/H + exchange in plasma membrane as it does for the purified Ca 2+-ATPase in proteoliposomes, indicating that measured Ca 2+/H + exchange activity is mainly due to a plasma membrane Ca 2+ pump. These suggest that calcium (Ca 2+) is transported out of Arabidopsis cells mainly through a Ca 2+-ATPase-mediated Ca 2+/H + exchange system that is driven by the proton-motive force from the plasma membrane H +-ATPase. 相似文献
12.
Protoplasts and vacuoles were isolated from immature apple fruit( Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca 2+uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent Km for Ca 2+ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa 2+ transport by protoplast and tonoplast vesicles, respectively.Ca 2+ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca 2+ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanide m-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca 2+ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent.
1Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986) 相似文献
13.
On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca 2+-ATPase is selectively extracted while approximately half of the initial Mg 2+-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca 2+-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg 2+-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca 2+-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg 2+-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca 2+-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures. 相似文献
14.
The GTP-driven component of Ca 2+ uptake in red beet ( Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+-translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45Ca 2+-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+-ATPases present in animal cells. 相似文献
15.
The vacuolar pH and the trans-tonoplast ΔpH modifications induced by the activity of the two proton pumps H +-ATPase and H +-PPase and by the proton exchanges catalyzed by the Na +/H + and Ca 2+/H + antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the 31P NMR technique. The H +-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast ΔpH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H +-ATPase. A ratio of H + translocated/ATP hydrolyzed of 1.97 ± 0.06 (mean ± standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H +-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H +-ATPase, building a steady state ΔpH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na + were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using 23Na NMR that sodium uptake was coupled to the H + efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca 2+/H + antiport has been measured on isolated intact vacuoles. Ca 2+ uptake was strongly inhibited by NH 4Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca 2+ were alkalinized by about 0.6 pH unit and this H + efflux was associated to a Ca 2+ uptake as demonstrated by measuring the external Ca 2+ concentration with a calcium specific electrode. Steady state accumulation ratios of Ca 2+ as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca 2+ uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca 2+/H + antiport has a high potential capacity to regulate cytoplasmic concentration of calcium. 相似文献
16.
Vanadate is a potent inhibitor of the Ca 2+-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca 2+ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca 2+Mg 2+-ATPases of the ascites and human red cell plasma membranes, Mg 2+-ATPase of the ascites plasma membrane, and the K +-ATPases of and hog gastric mucosal cell membranes. The ascites plasma membrane Ca 2+-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate. 相似文献
17.
The effects of NO 3− and assay temperature on proton translocating ATPases in membranes of barley ( Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H +-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO 3 when assayed at 24°C or above and was tentatively identified as the tonoplast H +-ATPase. A smaller peak of H +-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO 3 and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H +-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO 3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO 3. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO 3 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO 3 was explained by the temperature-dependent delay of the inhibition by KNO 3. The plasma membrane H +-ATPase maintained a pH gradient in the presence of KNO 3 for up to 30 minutes at 24°C. 相似文献
18.
The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca 2+-ATPase forms monomer-type (P1) type crystals in the E 1 and dimer-type (P2) crystals in the E 2 conformation. The P1 type crystals are induced by Ca 2+ or lanthanides; P2 type crystals are observed in Ca 2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca 2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca 2+-ATPase in the E 2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A 2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5′-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca 2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form. 相似文献
19.
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
20.
Microsomal membrane vesicles isolated from goat spermatozoa contain Ca 2+-ATPase, and exhibit Ca 2+ transport activities that do not require exogenous Mg 2+ .The enzyme activity is inhibited by calcium-channel inhibitors, e.g. verapamil and diltiazem, like the well known Ca 2+ , Mg 2+-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca 2+ can be completely released by the Ca 2+ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out 相似文献
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