胡杨液泡膜微囊的纯化及其质子转运活性

 为进一步研究液泡膜及 H+ - ATP酶在胡杨抵御盐胁迫中所起的作用 ,比较了研磨、捣碎和超声破碎三种细胞破碎方法 ,从悬浮培养的胡杨细胞中制备液泡膜微囊的效果 ;并用差速离心和不连续蔗糖密度梯度离心纯化了胡杨液泡膜微囊 .通过测定 H+ - ATP酶对 NO-3 、VO3-4和 Na N3的敏感性 ,以及焦磷酸酶质子转运活性表明 ,液泡膜微囊主要分布在 0 %～ 2 5%的蔗糖界面上 .捣碎法破碎细胞结合差速离心和蔗糖密度梯度离心可获得正向微囊比例高、封闭性好和酶活性高的液泡膜微囊     

Effects of pH on proton transport by vacuolar pumps from maize roots

Protons pumps of the tonoplast may be involved in the regulation of cytosolic pH, but the effects of pH on the coupled activities of these transporters are poorly understood. The effects of pH on the activities of the H⁺-translocating pyrophosphatase (PP_iase) and vacuolar-type H⁺-translocating adenosine triphosphatase (H⁺-ATPase) from maize ( Zea mays L. cv. FRB 73) root membranes were assessed by model that simultaneously considers proton transport by the pump and those processes that reduce net transport. The addition of either pyrophosphate or ATP to either microsomal or tonoplast membranes generated a pH gradient. The pH gradient generated in the presence of both substrates was not the sum of the gradients produced by the two substrates added separately. When membranes were separated by sucrose density gradient centrifugation, pyrophosphate (PP_i)-dependent proton transport was associated with light density membranes having tonoplast H⁺-ATPase activity. These results indicate that some portion of the PP_iase was located on the same membrane system as the tonoplast ATPase; however, tonoplast vesicles may be heterogeneous, differing slightly in the ratio of ATP- to PP_i-dependent transport. Proton transport by both the PP_iase and ATPase had maximal activity at pH 7.0 to 8.0 Decreases in proton transport by the ATPase at pH above the optimum were associated with increases in the processes that reduce net transport. Such an association was not observed at pH values below the optimum. These results are discussed in terms of in situ regulation of cytoplasmic pH by the two pumps.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Studies on the Proton Pumping Activity of H+-ATPase in Tonoplast Vesicles of Populus euphratica

Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton pumping activity of H+-ATPases in tonoplast vesicles were studied by acridine orange fluorescent quenching measured at 22 ℃. The proton pumping activity of ATPase was ATP-dependent with apparent Michaelis-Menten Constant (Km) for ATP about 0.65 mmol/L. The optimal pH for H+-ATPases activity was 7.5. The proton pumping activity of H+-ATPase could be initiated by some divalent cations, Mg2+ being highly efficient, much more than Fe2+; and Ca2+, Cu2+ and Zn2+ were inefficient under the experimental condition. The proton translocation could be stimulated by halide anions, with potencies decreasing in the order Cl-> Br->I->F-. The proton pumping activity was greatly inhibited by N-ethylmaleimide (NEM), N,N′-dicyclohexylcarbodiimide (DCCD), NO-3 and Bafilomycin A1, but not by orthovanadate and azide. These results demonstrated that the H+-ATPase in the tonoplast of Populus euphratica belonged to vacuolar type ATPase. This work was the first time that tonoplast-enriched vesicles were isolated from Populus euphratica cells.     

Tonoplast vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (T_A and T_B) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (T_B) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (T_A) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation.     

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO₃⁻-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg²⁺, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport

The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

separation and Immunological Characterization of Membrane Fractions from Barley Roots

Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross-reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72-kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two-dimensional gels of the PM and TG proteins.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Isolation of tonoplast vesicles from tobacco protoplasts

Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [³H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane.     

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

The control by delta mu H+ of the tonoplast-bound H+-translocating adenosine triphosphatase from rubber-tree (Hevea brasiliensis) latex.

The relationship between tonoplast-bound ATPase activity and the magnitude of the electrochemical proton gradient has been investigated on tightly sealed vesicles prepared from rubber-tree (Hevea brasiliensis) latex. A variety of methods have been used to modify, either alone or together, the two components of the electrochemical proton gradient (delta mu H+). When the delta pH component was decreased either by titration with (NH4)2SO4 or by addition of protonophores or nigericin in the presence of K+, ATPase activity was stimulated. On the other hand, when the delta psi component was decreased either by addition of lipophilic cations or by addition of valinomycin in the presence of K+, ATPase activity decreased. It is concluded that activity of the tonoplast-bound ATPase is regulated by changes in the electrochemical proton gradient across the tonoplast, so that, once the maximum proton gradient is established across the tonoplast, any perturbation of the equilibrium state should result in the increased rate of ATP hydrolysis as the enzyme attempts to re-establish the initial gradient.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Localization of a proton-translocating ATPase on sucrose gradients

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing β-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.     

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H⁺ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H⁺ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I₅₀ = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H⁺ pumping activities. This is in contrast to the H⁺-PPase of higher plants which is 4,4′-stilbenedisulfonic acid insensitive.     

NaCl胁迫对高粱根、叶鞘和叶片液泡膜ATP酶和焦磷酸酶活性的影响

NaCl胁迫初期 ,Na 主要在根和叶鞘中积累。相应地 ,根和叶鞘液泡膜ATP酶和焦磷酸酶水解活性、依赖ATP和PPi的质子泵活性及Na /H 逆向转运活性均明显增加 ,根和叶鞘的生长没有受到抑制。NaCl胁迫后期 ,Na 开始向地上部分运输并在叶片中积累。此时 ,叶片液泡膜质子泵和Na /H 逆向转运活性开始增加 ,根和叶鞘的Na/K比增加 ,其液泡膜ATP酶和焦磷酸酶水解活性、质子泵活性和Na /H 逆向转运活性下降。相应地 ,根和叶鞘的生长也下降。当保温介质中Na/K比超过 1时 ,液泡膜微囊ATP酶和焦磷酸酶活性均随Na/K比的增加而下降。表明非盐生植物液泡膜质子泵在盐胁迫的初期对Na 在液泡内的积累及其耐盐性起重要作用