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1.
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
2.
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
3.
Corn ( Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H +-transport as measured by [ 14C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide- m-chlorophenylhydrazone. Of the divalent cations tested, Mn 2+ is almost as effective as Mg 2+, while Ca 2+ is ineffective. Excess divalent cations, particularly Ca 2+, reduces the pH gradient. H + transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with 36Cl − indicate that ATP-driven H + transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide- m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and 36Cl − uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin. 相似文献
4.
The Ca 2+ transport system of corn ( Zea mays) leaf plasma membrane is composed of Ca 2+ pump and Ca 2+/H + antiporter driven by H + gradient imposed by a H + pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca 2+ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca 2+ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C 12E 8) was the most effective detergent for a series of extraction and functional reconstitution of the Ca 2+ pump among seven detergents examined. This was judged from activities of ATP-dependent 45Ca 2+ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C 12E 8-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca 2+-ATPase was separated from VO 43−-sensitive Mg 2+-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca 2+ uptake was measured. The liposomes reconstituted with the Ca 2+-ATPase, but not with the VO 43−-sensitive Mg 2+-ATPase, showed ATP-dependent Ca 2+ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca 2+ uptake into liposomes reconstituted with the Ca 2+-ATPase, suggesting that the Ca 2+/H + antiporter was not present in the preparation. These results indicate that the Ca 2+-ATPase actually functions as Ca 2+ pump in the corn leaf plasma membrane. 相似文献
5.
Absorption characteristics of Mg 2+ and Cl − were investigated with 5-day-old excised corn ( Zea mays) roots. Uptake from both 0.5 and 10 milliequivalents per liter MgCl 2 solutions occurred at steady state rates for the first 6 hours. Inhibition by dinitrophenol and low temperatures established that absorption during this period was metabolically mediated in the absence and presence of Ca 2+. Absorption isotherms indicated dual mechanisms of Mg 2+ and Cl − absorption from solutions above 1 milliequivalent per liter. The effect of H + on absorption of Mg 2+ and Cl − was typical of that generally reported for other plant roots and other ions. In the physiological pH range, Ca 2+ greatly suppressed the rate of Mg 2+ absorption but had little effect on Cl −. The influence of Ca 2+ on Mg 2+ appeared to be noncompetitive and independent of its effect on membrane permeability. 相似文献
6.
The presence of Ca 2+ ions in solution is vital for root growth. The plasma membrane is one of the first sites where competition between Ca 2+ and other ions occurs. We studied the competition between Ca 2+ and Na + or Mg 2+ for sorption sites on the plasma membrane of melon root cells.Sorption of 45Ca 2+ to right-side-out PM vesicles of melon ( Cucumis melo L.) roots (prepared by aqueous two-phase partitioning) was studied at various Ca 2+ concentrations, in the presence of increasing concentrations of Na + or Mg 2+ chlorides. Experimentally determined amounts of Ca 2+ sorbed to the plasma membrane vesicles agreed fairly well with those calculated from a competitive sorption model. The best fit of the model to the experimental data was obtained for an average surface area of 370 Å 2 per charge, and binding coefficients for Na +, Mg 2+ and Ca 2+ of 0.8, 9 and 50 m
-1, respectively.Our results suggest that nonphospholipid components in the plasma membrane contribute significantly to Ca 2+ binding. The high affinity of Ca 2+ binding to the plasma membrane found in this study might explain the specific role of Ca 2+ in relieving salt stress in plant roots.This research was supported by the GIFRID German-Israel fund for research and international development. 相似文献
7.
The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg 2+-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg 2+-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg 2+- or Ca 2+-dependent ATPases were associated with the plasma membrane. Ca 2+-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca 2+ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg 2+-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H +-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca 2+. The reduced fluorescence was recovered with the addition of 10 mmol/L NH 4Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca 2+-dependent ATPase might pump H + out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification. 相似文献
8.
We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca 2+ or Mg 2+ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90° light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca 2+ and Mg 2+ at threshold concentration ranges of 0.03–0.1 mM (Ca 2+) and 0.07–0.15 mM (Mg 2+) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca 2+ or Mg 2+ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5–10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion. 相似文献
9.
Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K +, along with small but significant loss of cellular Ca 2+. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca 2+ from membrane by extracellular K + and subsequent perturbation of K + transport properties of plasma membrane. In the present study, onion ( Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca 2+ compared to control. Loss of membrane-associated Ca 2+ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca 2+-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl 2. Our results suggest that the loss of membrane-associated Ca 2+, in part, plays a role in initiation and progression of freezing injury. 相似文献
10.
Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat ( Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP 2 phospholipase C was dependent on Ca 2+ with maximum activity at 10 to 100 μ m free Ca 2+ and half-maximal activation at 0.1 to 1 μ m free Ca 2+. In the presence of 10 μ m Ca 2+, 1 to 2 m m MgCl 2 or MgSO 4 further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 m m Ba 2+, Co 2+, Cu 2+, Mn 2+, Ni 2+, and Zn 2+) inhibited the enzyme activity. The stimulatory effect by Mg 2+ was observed also when 35 m m NaCl was included. Thus, the PIP and PIP 2 phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP 2 as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca 2+, with maximum activity at 1 m m CaCl 2, and could not be further stimulated by Mg 2+. 相似文献
11.
ATPase activity of plasma membranes isolated from oat ( Avena sativa L. cv. Goodfield) roots was activated by divalent cations (Mg 2+ = Mn 2+ > Zn 2+ > Fe 2+ > Ca 2+) and further stimulated by KCl and a variety of monovalent salts, both inorganic and organic. The enzyme exhibited greater specificity for cations than anions. The presence of Mg 2+ was necessary for KCl stimulation. Ca 2+ was ineffective in replacing Mg 2+ for activation of plasma membrane ATPase, but it did activate other membrane-bound ATPases. The pH optima for Mg 2+ activation and KCl stimulation of the plasma membrane ATPase were 7.5 and 6.5, respectively. 相似文献
12.
The protein-sensitized fluorescence of Tb 3+ was used as a probe for cation binding sites on synaptic vesicles. Competition studies show that the order of affinity for the sites is Cu 2+ > Mn 2+ > Ca 2+ > Mg 2+ and Zn 2+ is inactive. Fluorescence quenching studies indicate that the site is superficial and the effect of pH suggests that histidine is involved in the binding. Measurements of enzyme activities in the presence of lanthanides reveal that the metal binding site identified by Tb 3+ fluorescence is not the Cu 2+ site associated with dopamine-β-hydroxylase. Terbium inhibits Ca 2+-stimulated ATPase but not Mg 2+-stimulated ATPase activities of the synaptic vesicle fraction. A kinetic analysis indicates that the site monitored by Tb 3+ fluorescence may be a component of the Ca 2+-stimulated ATPase. It is also suggested that Mg 2+ and especially Cu 2+ may bind to the sites in vivo, serving as a bridge between vesicles and other synaptic components such as the presynaptic plasma membrane. 相似文献
13.
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca 2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca 2+ transport was assayed by membrane filtration technique. Results showed that Ca 2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca 2+ transport system had a high affinity for Ca 2+( K
m
(Ca 2+)=0.4 m) and ATP( K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca 2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl – or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H + transport into the vesicles. The Ca 2+ uptake stimulated by Cl – was dependent on the activity of H + transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H + pump associated with the plasma membrane, canceled almost all of the Cl –-stimulated Ca 2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca 2+ uptake into the vesicles. These results suggest that the Cl –-stimulated Ca 2+ uptake is caused by the efflux of H + from the vesicles by the operation of Ca 2+/H + antiport system in the plasma membrane. In Cl –-free medium, H + transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca 2+ uptake into the vesicles. These results suggest that two Ca 2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca 2+ transport system (Ca 2+ pump) and the other is a Ca 2+/H + antiport system. Little difference in characteristics of Ca 2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
14.
Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet ( Beta vulgaris L.) were used to study the kinetics of a Ca 2+/H + antiport across this membrane. Ca 2+-dependent H + fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca 2+ influx was measured radiometrically. Both H + efflux and Ca 2+ influx displayed saturation kinetics and an identical dependence on external calcium with apparent Km values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg 2+ but was inhibited by La 3+ > Mn 2+ > Cd 2+. The apparent Km for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested. 相似文献
15.
Summary The plasma membrane (Mg 2+)-dependent adenosine triphosphatase ((Mg 2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg 2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H + transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca 2+)-dependent adenosine triphosphatase ((Ca 2+)-ATPase) activity. Proton efflux associated with (Ca 2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca 2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982) J. Biol. Chem.
257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg 2+)-ATPase to pump 28Mg 2+, 35SO
4
2–
and 86Rb + was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system. 相似文献
16.
Salinity-induced alterations in tomato ( Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H +-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants ( Kd) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured Kd values and the calculated intrinsic affinity constant, Ki. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K +-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl − ions to stimulate H +-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane. 相似文献
17.
Bundle sheath strands were isolated from maize ( Zea mays L.) leaves treated with preparations of cellulase, hemicellulase, and pectinase. A three-phase discontinuous gradient yielded two fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm −3. The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of electron microscopy. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic, and electron microscopic examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes, and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56-g leaf sections. The Mg 2+-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 μmoles Pi released hr −1 mg protein −1, a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants. 相似文献
18.
Membrane fusion induced by Ca 2+ and Mg 2+ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca 2+ or Mg 2+ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca 2+ or Mg 2+. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca 2+ in the presence of a Mg 2+ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca 2+, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H II) transition in membrane fusion, was examined by the temperature dependence of Ca 2+-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes. 相似文献
19.
A previous study of energy-independent in vitro Ca 2+ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH) 2D 3 repletion ( R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca 2+ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg 2+ and Sr 2+, but not by Na + or K +. Lowering the external [H +] or raising the internal [H +] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H +] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl 2 or SrCl 2 but not CaCl 2, NaCl, or KCl. Vesicles preloaded with K 2SO 4 failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl 2 was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca 2+ uptake maintained at equilibrium. Therefore, the remainder of the Ca 2+ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10 ?6m was found for this binding. The bound Ca 2+ could be rapidly released by external Mg 2+ or Sr 2+, but not Ca 2+, Na +, or K +. Release of bound Ca 2+ could also be induced by raising the [H +] of the external medium. Failure of external Ca 2+ to release bound Ca 2+ suggested that the release induced by external Mg 2+, Sr 2+, or H + was not due to competitive displacement of Ca 2+ from its binding sites. These results indicated that Ca 2+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca 2+ to the vesicle. The effects of H +, Mg 2+, and Sr 2+ on Ca 2+ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles. 相似文献
20.
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
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