磷饥饿下番茄幼苗根系液泡膜H+-ATPase活性的适应性变化

以‘世纪星’番茄为材料。研究了磷饥饿下番茄幼苗的生长状况及其根部液泡膜H^＋-ATPase活性的适应性变化。结果表明：磷饥饿下番茄幼苗的平均高度均低于对照苗,而主根长度均显著长于对照。磷饥饿提高了番茄幼苗根部液泡膜H^＋-ATPase的水解活性,随着磷胁迫时间的延长,该酶的活性逐渐增大,在磷饥饿7d时达到最大,后又略有降低;而对照番茄幼苗根部该酶的活性变化很小。动力学分析表明：磷饥饿使番茄幼苗根部液泡膜H^＋-ATPase的Km值明显降低,但对该酶的Kmax影响不大。这说明磷饥饿提高了该酶对其底物的亲和力。此外,磷饥饿并不改变液泡膜H^＋-ATPase酶的最适pH值（仍为7．5）。     

盐地碱蓬叶片液泡膜H^＋—ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H^ -ATPase在建立跨液泡膜质子梯度、促进液泡Na^ 区域化、提高植物耐盐性方面发挥着重要作用。本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H^ -ATPase B亚基cDNA克隆。测序表明该基因长达1974bp,开放阅读框有1470bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54．29kD。Northern及Western印迹表明盐地碱蓬液泡膜H^ -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H^ -ATPase c亚基存在协同作用。盐胁迫下,盐地碱蓬液泡H^ -ATPase B亚基与c亚基的协同表达增加了液泡H^ -ATPase的数量,从而提高了液泡H^ -ATPase活性,为碱蓬叶片液泡Na^ 区域化提供了动力,最终提高了碱蓬植株的耐盐性。     

盐胁迫对豌豆根液泡膜H^＋—ATPase活性及含量的影响

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性调节机制,对豌豆（Pisum sativum L.）植株进行不同盐浓度和不同盐胁迫时间（1－3d）的处理后,分别测定液泡膜H^ -ATPase的H^ 转运活性、水解性和蛋白含量（A亚基）的变化。结果表明,100mmol/L和200mmol/L NaCl 处理1dH^ -ATPase的水解活性没有变化,而250mmol/L NaCl处理1d引起水解活性降低约25％。100mmol/L NaCl处理2d内水解活性没有变化,而第3天活性下降约20％。但是上述盐胁迫均能提高液泡膜H^ -ATPase的质子转运活性,说明盐胁迫后H^ -ATPase的水解活性和质子转运活性的变化不成比例,盐胁迫可能导致偶联比率的改变。Western blot研究发现,上述盐胁迫对液泡膜H^ -ATPase（A亚基）的含量基本无影响,仅100mmol/L NaCl处理3d后A亚基的量略有下降,这些结果证明,盐胁迫能刺激提高豌豆根液泡膜H^ -ATPase的H^ 泵效率,且泵效率的提高是源于偶联比率的改变,而不是由于ATP水解活性的提高和蛋白含量的增加。     

嫁接的西瓜果实发育过程中叶和果实蔗糖代谢的一些特性

嫁接瓜果实发育过程中,叶内蔗糖含量和干物质积累显著高于自根瓜,自根瓜和嫁接瓜的叶中蔗糖含量与酸性转化酶（AI）、蔗糖磷酸合酶（SPS）活性均呈显著正相关;嫁接瓜果实中蔗糖含量显著低于自根瓜,SPS活性和果实中糖分的跨液泡膜运输能力亦较自根瓜低;自根瓜和嫁接瓜叶的干物质积累与叶中AI和SPS活性、果实AI活性呈显著负相关,与液泡膜H^＋-ATPase活性呈显著正相关。     

NaCl胁迫下大麦根系液包膜H^＋—ATPase活性与膜流动性的关系

用50－200mmol/L NaCl处理2d后,大麦（Hordeum vulgare L.)品种“滩引2号”（耐盐性强）根的液泡膜H^ -ATPase活性增强,600mmol/L NaCl处理下酶活性下降;“科品7号”（耐盐性弱）在50－100mmol/L NaCl处理2d后根的液泡膜H^ -ATPase活性增强,200－600mmol/L NaCl处理下酶活性随盐浓度增加而降低。50－200mol/L NaCl处理下“滩引2号”根的液泡膜流动性下降,600mmol/L NaCl处理下膜流动性明显增大;盐胁迫下液泡膜膜脂脂肪酸不饱和度下降时,膜流动性下降,反之则膜流动性上升。由此推断高盐胁迫下液泡膜膜脂脂肪酸不饱和度上升而引起膜流动性上升可能是引起H^ -ATPase活性下降的原因之一。     

灵武长枣果实质膜和液泡膜H~+-ATPase和H~+-PPase活性与果实糖分积累

以不同发育时期灵武长枣(Ziziphus jujuba cv.Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜H+-ATPase和H+-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜H+-ATPase和H+-PPase活性与糖积累特性的关系。结果表明:(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖。(2)在果实发育的缓慢生长期S1,质膜H+-ATPase活性最低;第一次快速生长期,质膜H+-ATPase活性最高;缓慢生长期S2,其活性降低;第二次快速生长期,质膜H+-ATPase活性升至次高;完熟期,质膜H+-ATPase活性下降幅度较大。(3)在果实发育过程中,液泡膜H+-ATPase和H+-PPase活性的变化趋势相似。缓慢生长期S1,液泡膜H+-ATPase和H+-PPase活性较低;从缓慢生长期S1至第一次快速生长期缓慢下降至最低;从第一次快速生长期开始,液泡膜H+-ATPase和H+-PPase活性呈现为逐渐增高的变化趋势;除第二次快速生长期以外,液泡膜H+-PPase活性始终高于H+-ATPase。由此推测,质膜H+-ATPase和液泡膜H+-ATPase、H+-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用。     

K^＋营养对NaCl胁迫下盐地碱蓬生长及叶片液泡膜V—H^＋—ATP和V—H^＋—PPase活性的影响

对溶液培养的盐地碱蓬（Suaeda salsa L.)幼苗进行不同浓度NaCl胁迫并改变培养液中K^ 浓度,以了解K^ 营养对NaCl胁迫下盐地碱蓬幼苗生长及叶片液泡膜V－H^ -ATPase、V－H^ -PPase活性的影响。提高培养液K^ 浓度可明显增加盐胁迫下碱蓬植株的鲜重、干重,促进盐地碱蓬叶片及根部组织K^ 积累。盐地碱蓬叶片液泡膜V－H^ -ATPase至少由A、B、C、D、E及c亚基组成,其表达量在缺K^ 处理（12μmol/L K^ )下随盐胁迫浓度的增加而减小,而在正常K^ （6mmol/L)培养下则随盐胁迫浓度的增加而增加;盐地碱蓬叶片液泡膜V－H^ -PPase分子量为72kD,在缺K^ 和正常K^ 供应情况下,V－H^ -PPase均有较高表达。V－H^ -ATPase及V－H^ -PPase活性变化与其亚基表达量变化基本成正相关。结果表明：K^ 对盐生植物碱蓬的耐盐性有重要作用,盐胁迫下,K^ 可能参与了V－H^ -ATPase和V－H^ -PPase活性调控。     

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     

Na+/H+逆向转运蛋白和植物耐盐性

Na^ /H^ 逆向转运蛋白对植物耐盐起着重要作用,它利用质膜H^ -ATPase或液泡膜H^ -ATPase及PPiase泵H^ 产生的驱动力把Na^ 排出细胞或在液泡中区隔化以消除Na^ 的毒害。主要讨论植物中Na^ /H^ 逆向转运蛋白研究在分子水平的最新进展。     

钙对盐胁迫下棉苗离子吸收分配的影响

研究了钙对NaCl胁迫下棉花幼苗体内离子分布的影响及其与根系质膜H^ -ATP酶、液泡膜H^ -dATP酶和H^ -PP酶活性的关系。不同器官离子含量和根系横切面X-射线微区分析结果表明,NaCl胁迫下外源钙明显减少棉花幼苗对Na^ 的吸收及其向茎杆、叶片的运输,增加对K^ 和Ca^2 的吸收及其向茎相杆、叶片的运输,增强棉苗体内的盐分区域化分配,提高根冠比和干物质积累,根系电解质渗漏率下降,钙明显提高盐胁迫下幼根细胞质膜H^ -ATP酶、液泡膜H^ -ATP酶和H^ -PP酶的活性,与钙调节棉花对离子的吸收、分配相一致,说明这些酶可以为根细胞中的Na^ 在液泡中积累以及K^ 、Ca^2 的选择性吸收和运输提供动力。     

外源海藻糖对小麦幼苗耐盐性的影响

以盐敏感小麦品种鲁麦15为材料,分别用完全Hoagland营养液、150mmol／L NaCl和150mmol／L NaCl 10mmol／L海藻糖处理小麦幼苗,测定小麦幼苗生长、离子含量、根系质膜H^ -ATPase、SOD活性、MDA含量等指标,旨在探讨外源海藻糖在抗盐性中的作用。结果表明：外源海藻糖可明显缓解盐胁迫对小麦幼苗生长的抑制作用;明显提高NaCl胁迫条件下小麦幼苗叶片中K^ 的含量,降低Na^ 的含量,降低其Na^ ／K^ ;提高NaCl胁迫条件下小麦幼苗SOD活性,降低MDA的含量,降低细胞质膜透性,缓解根系质膜H^ -ATPase活性抑制。以上结果表叫外源海藻糖可能通过增加活性氧清除能力、缓解质膜伤害、维持胞质离子稳态提高植物抗盐性。     

The leaf tonoplast V-H(+)-Atpase activity of a C3 halophyte Suaeda salsa is enhanced by salt stress in a Ca-dependent mode

Suaeda salsa seedlings grown in Hoagland nutrient solution were treated with different concentrations of NaCl combined with two levels of Ca2+ (0 and 20 mmol/L) to study the effect of Ca2+ nutrition on the growth and activity of leaf tonoplast V-H(+)-ATPase. Increase of Ca2+ concentration in the solution markedly increased the relative growth quantity of S. salsa seedlings and Ca2+ and K+ concentration in the leaf cell sap under NaCl stress. The leaf V-H(+)-ATPase activity was significantly increased with increasing NaCl concentration under high Ca2+ application (20 mmol/L), but little changed under Ca2+ starvation (0 mmol/L). Western blot analysis showed that the leaf V-H(+)-ATPase of S. salsa was at least composed of A, B, D and c subunits, and their protein amounts were not affected by NaCl treatments under Ca2+ starvation (0 mmol/ L) with an exception of 100 mmol/L NaCl, but increased under high Ca2+ application (20 mmol/L). There was a positive correlation between activity of V-H(+)-ATPase and the protein amounts of the subunits. The results suggest that Ca2+ nutrition played an important role in the salt tolerance of S. salsa, and that enhancement of V-H(+)-ATPase activity under salt stress was Ca2(+)-dependent.     

Protective Effects of Glycinebetaine on Brassica chinensis Under Salt Stress (in English)

Brassica chinensis L. were foliarly applied with glycinebetaine (GB), as this species is unable to synthesis GB and sensitive to osmotic stress such as salt. The exogenous GB was easily absorbed and transported by the leaf of B. chinensis . Its application (0-20 mmol/L) enhanced the plant tolerance to salt stress. The treatment of 15 mmol/L GB significantly decreased the Na+ accumulation in leaf and root under NaCl stress. This difference in accumulating Na+ and K+ is caused by higher selectivity of root absorption. Furthermore, GB increased H+-ATPase activity of root plasma membrane evidently. This result strongly suggested that in root the decreased Na+ accumulation was caused by the GB accumulation that enhanced the extrusion of Na+ from the cell in some way through plasma membrane transporter, e.g. Na+/H+ antiport driven by H+-ATPase. The GB application was also found to stabilize the plasma membrane, to decrease the loss of chlorophyll, and to stimulate the osmosis induced proline response under salt stress.     

Effects of Salt Stress on Carbohydrate Metabolism in Desert Soil Alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides （EPS） in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCI, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCI. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K＋ concentrations of stressed cells decreased significantly and H＋-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmOtic equilibrium between the intra-and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na＋ and K＋ by H＋-ATPase.     

水分胁迫对玉米幼叶生长区细胞质膜H~+-ATPase活性的影响

PEG/Hoagland(-0.1 MPa)溶液根际胁迫处理5叶期玉米幼苗24h,明显刺激第5幼叶生长区细胞H?的分泌,比对照高约1.5倍,钒酸钠对此过程有强烈抑制作用。用水溶性多聚物(PEG4000/Dextran T 500)两相分配法分离玉米第5幼叶生长区的质膜,胁迫处理增加了质膜H~+—ATPase对底物的亲和力,K_m降低到对照的?;活力增加约1.5倍。亚胺环己酮对胁迫处理引起的质膜H~+—ATP_(ase)活性增加没有抑制作用,不同程度的胁迫处理会导致膜制剂磷脂/蛋白比率的变化,其比率在0.83～1.05之间时.随比率的增加,质膜 H~+—ATP_(ase)活性迅速上升;比率在1.05～1.37之间时,随比率值增加,活性迅速下降。暗示质膜的磷脂含量变化可能是胁迫导致质膜 H~+—ATP_(ase)活性增加的主要原因。     

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.     

Effects of Salt Stress on the Activity and the Amount of Tonoplast H+-ATPase from Pea Roots

To study the function and adaptive mechanism of tonoplast H+ATPase under salt stress, pea ( Pisum sativum L.) seedlings were treated with different concentrations of salt （100-250 mmol/L NaCl） and with 100 mmol/L NaCl for different days (1-3 d). The ATP hydrolytic activity and the proton transport activity and the changes of the amount of tonoplast H+ ATPase (subunit A) were measured. ATP hydrolytic activity of H+ATPase prepared from plants treated with 250 mmol/L NaCl was reduced by about 25% compared to that of control plants, but that of stressed plants treated with 100 mmol/L and 200 mmol/L NaCl was unchanged. The activity from plants treated with 100 mmol/L NaCl for up to 3 d was lower than that of control plants by 20%. But the proton transport activity was increased under the same salt stresses as above. These results showed that the changes of the hydrolytic activity and the proton transport activity were not in proportion and salt stress may cause the change of the coupling ratio of H+ transport activity to ATP hydrolysis. The protein amount kept unchanged and reduced a little only when pea was treated with 100 mmol/L NaCl for 3 d. These results indicated that salinity stimulated the increase of the pump efficiency of the V-ATPase from pea roots, which was due to the change of the coupling ratio, but not due to the increase of ATP hydrolysis and the amount of V-ATPase.     

Basal-lateral membranes from rabbit renal cortex prepared on a large scale in a zonal rotor

Basal-lateral membranes from the renal cortex of the rabbit were isolated by sucrose gradient centrifugation in a zonal rotor which allows for a large-scale preparation of these membranes. A heterogeneous population of membranes (P4) which contained 29% of the (Na+ + K+)-ATPase found in the homogenate of renal cortex was prepared by differential centrifugation. When pellet P4 was subjected to centrifugation in a sucrose gradient the activity of (Na+ + K+)-ATPase, a marker for basal-lateral membranes, could be separated from enzymatic markers of other organelles. The specific activity of (Na+ + K+)-ATPase was enriched 12-fold at a density of 1.141 g/cm3. Membranes (P alpha) contained in the (Na+ + K+)-ATPase-rich fractions consisted primarily of closed vesicles which exhibited probenecid inhibitable transport of rho-aminohippurate. These membranes did not exhibit Na+-dependent, phlorizin-inhibitable D-glucose transport. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from P alpha revealed at least six major protein bands with molecular weights of 91000, 81000, 73000, 65000, 47000 and 38000. A small fraction of total alkaline phosphatase found in the homogenate was found in pellet P4. Membranes containing this alkaline phosphatase activity were distributed widely over the gradient, with peak activity found at a density of 1.141 g/cm3. In contrast, when brush borders were subjected to gradient centrifugation under the same conditions as P4, alkaline phosphatase was found in a narrow distribution, with peak activity at a density of 1.158 g/cm3. The principle subcellular localization of the alkaline phosphatase found in P4 could not be determined unambiguously from the data, but the activity did not seem to be primarily associated with classical brush borders.     

Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H