绿豆下胚轴质膜微囊的提取和H+-ATPase活性研究

以两相法提取纯化绿豆下胚轴质膜微囊,材料与两相体系重量之比为32:8时,一次洗膜就可以得到纯度较高的质膜微囊.提取缓冲液中牛血清白蛋白的浓度对质膜H -ATPase的潜在活性有影响.质膜H -ATPase水解活性依赖于Mg2 ,Ca2 对酶活性有明显的促进作用.壳梭孢素(fusicoccin,FC)对酶有明显的刺激作用,活体条件最大刺激达到72%,而离体条件下刺激为30%.     

绿豆下胚轴质膜微囊的提取和H+-ATPase 活性研究

以两相法提取纯化绿豆下胚轴质膜微囊,材料与两相体系重量之比为32∶8时,一次洗膜就可以得到纯度较高的质膜微囊。提取缓冲液中牛血清白蛋白的浓度对质膜H+-ATPase的潜在活性有影响。质膜H+-ATPase水解活性依赖于Mg2+,Ca2+对酶活性有明显的促进作用。壳梭孢素（fusicoccin, FC）对酶有明显的刺激作用,活体条件最大刺激达到72％,而离体条件下刺激为30％。     

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ATP酶对低温(8℃)及高pH(8.0)胁迫的反应。结果表明水稻根细胞质膜和液泡膜上均存在Ca3+-ATP酶,但活性远低于H+-ATP酶。耐冷品种武育粳3号经低温(8℃)处理2d,根系质膜和液泡膜H+-ATP酶、Ca2+-ATP酶活性均明显升高,至冷处理12d,H+-ATP酶、Ca2+-ATP酶活性有所下降,但仍与对照相近;而冷敏感品种汕优63经低温(8℃)处理2d,根系质膜H+-ATP酶活性略有升高,而质膜Ca2+-ATP酶以及液泡膜H+-ATP酶、Ca2+-ATP酶活性已明显下降;至冷处理12d,4种酶活性均明显低于对照。高pH胁迫使质膜和液泡膜H+-ATP酶活性下降,而使Ca2+-ATP酶活性上升。高pH胁迫会加剧低温冷害。结果表明,耐冷品种质膜、液泡膜ATP酶比冷敏感品种对低温胁迫有更强的适应能力。     

茉莉酸甲酯和脱落酸对绿豆下胚轴质膜H+-ATPase水解活性的影响

茉莉酸甲酯(MeJA)促进绿豆下胚轴质膜H+-ATPase水解活性.活体条件下,50μmol·L-1 MeJA处理7 h的酶活性提高30%;离体条件下,10 μmol·L-1 MeJA处理2 h的酶活性最大,即提高30%.壳梭孢素(FC)和MeJA在离体条件下对H+-ATPase活性的促进效应相同,均提高30%左右,无协同效应;活体条件下,FC促进质膜H+-ATPase水解活性可达70%,而MeJA仅为30%.离体条件下,脱落酸(ABA)对H+-ATPase水解活性无明显促进;而活体条件下则有一定的抑制.     

大豆叶片衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化

以大豆幼苗初生叶为材料研究了衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化。结果发现质膜上一个57kD的蛋白激酶分子上有多个自磷酸化位点,而且自磷酸化反应能提高该酶催化组蛋白H1磷酸化的激酶活力。进一步的研究表明诱导衰老处理造成的57kD蛋白激酶自磷酸化状态的变化,可能对调节它在衰老过程中催化活性的变化起重要作用;而外源6-BA预处理则能够维持57kD蛋白激酶体内高自磷酸化状态,保持该激酶在衰老过程中的催化活力。对衰老和6-BA处理过程中质膜上39和47kD蛋白激酶自磷酸化状态变化的研究表明,这两种激酶可能参与大豆叶片对6-BA刺激信号的传导和/或应答反应过程。     

大豆叶片衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化

以大豆幼苗初生叶为材料研究了衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化,结果发现质膜上一个57kD的蛋白激酶分子上有多个自磷酸化位点,而且自磷酸化反应能提高该酶催化组蛋白H1磷酸化的激酶活力。进一步的研究表明诱导衰老处理造成的57kD蛋白激酶自磷酸化状态的变化,可能对调节它在衰老过程中催化活性的变化起重要作用;而外源6－BA预处理则能够维持57kD蛋白激酶体内高自磷酸化状态,保持该激酶在衰老过程中的催化活力。对衰老和6－BA过程中质膜上39和47kD蛋白激酶自磷酸化状态变化的研究表明,这两种激酶可能参与大豆叶片对6－BA刺激信号的传导和／或应答反应过程。     

外源钙和茉莉酸甲酯诱导番茄植株抗灰霉病研究

以‘辽园多丽’番茄幼苗为材料,研究了经钙(Ca)、钙螯合剂(EGTA)和茉莉酸甲酯(MeJA)处理后接种番茄灰霉病幼苗叶片的病情指数、活性氧(H2O2、O2.-)含量和过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性的变化。结果显示:(1)Ca、MeJA、MeJA+Ca处理番茄幼苗的灰霉病发病率分别比对照显著降低32.5%、38.0%和54.5%,而MeJA+Ca处理又显著低于Ca、MeJA处理32.6%和15.3%;MeJA+EGTA处理高于MeJA处理30.3%,但低于EGTA处理13.1%;Ca处理低于EGTA处理34.2%。(2)Ca、MeJA及MeJA+Ca处理番茄幼苗叶片中活性氧积累量高于对照,MeJA+Ca处理又高于Ca、MeJA处理;但MeJA+EGTA处理活性氧积累量低于MeJA处理,而高于EGTA处理;Ca处理的活性氧含量高于EGTA处理。(3)Ca、MeJA及MeJA+Ca处理幼苗叶片的SOD、CAT、POD的活性均比对照提高,且以MeJA+Ca处理最高;而MeJA+EGTA处理抗氧化酶活性低于MeJA处理,但高于EGTA处理;Ca处理抗氧化酶活性高于EGTA处理。研究表明,钙在茉莉酸甲酯诱导番茄抗灰霉病过程中具有重要调节作用,这种作用与钙促进茉莉酸甲酯诱导番茄活性氧积累和抗氧化酶活性有关。     

水稻幼苗根细胞质膜和液泡膜微囊Ca2+-ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2 -ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2 -ATP酶活性,但促进液泡膜Ca2 -ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

水稻幼苗根细胞质膜和液泡膜微囊Ca^2＋－ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2+-ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2+-ATP酶活性,但促进液泡膜Ca2+-ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

抗寒剂CR-4 对冬小麦幼苗质膜钙泵（Ca2 +-ATPase） 的稳定作用

通过磷酸铈沉淀的细胞化学观察揭示,常温下生长的冬小麦幼苗的Ca2+ -ATP酶活性主要定位在质膜上,同时,水浸种和抗寒剂浸种的小麦质膜Ca2+ -ATP酶活性没有差异。然而,小麦幼苗经-7℃冰冻处理12小时和24小时后,则表现明显的区别：水浸种的小麦幼苗质膜Ca2+ -ATP酶活性明显下降,直至完全失活,细胞的精细结构也同时被破坏;而经抗寒剂浸种的小麦幼苗质膜Ca2+ -ATP酶仍维持较高的活性,细胞结构也保持完整,显示抗寒剂对质膜Ca2+ -ATPase酶起着明显的稳定作用。     

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.     

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

The effect of controlled proteolysis on the plasma membrane (PM)Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125l-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase co-migrates with the phosphorylated intermediate of the enzyme[mdash]labeled by incubation with [[gamma]-32P]GTP in the presence of calcium[mdash]on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Modulation of reactive oxygen species production during osmotic stress in Arabidopsis thaliana cultured cells: involvement of the plasma membrane Ca2+-ATPase and H+-ATPase

In Arabidopsis thaliana cells, hypoosmotic treatment initially stimulates Ca2+ influx and inhibits its efflux and, concurrently, promotes a large H2O2 accumulation in the external medium, representative of reactive oxygen species (ROS) production. After the first 10-15 min, Ca2+ influx rate is, however, lowered, and a large rise in Ca2+ efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca2+, are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca2+-ATPase of the plasma membrane (PM). Comparable changes of Ca2+ fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca2+ efflux is activated. Also K+ and H+ net fluxes across the PM and cytoplasmic pH (pH(cyt)) are very differently influenced by the two opposite stresses: strongly decreased by hypoosmotic stress and increased under hyperosmotic treatment. The H2O2 accumulation kinetics, followed as a function of the pH(cyt) changes imposed by modulation of the PM H+-ATPase activity or weak acid treatment, show a close correlation between pH(cyt) and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca2+-ATPase in switching off the signal triggering ROS production, and propose a role for the PM H+-ATPase in modulating the development of the oxidative wave through the pH(cyt) changes following the changes of its activity induced by stress conditions.     

N-Ethylmaleimide Modifies the Conformation of the Plasma Membrane H+-ATPase,Strengthening the Inhibitory Action of the C-terminal Domain1

Abstract: The effect of cysteine modification with N-ethylmalei-mide (NEM) on the activity of the plasma membrane (PM) H+-ATPase and on its activation state was investigated in PM isolated from aged red beet parenchyma slices. Treatment of PM with increasing concentrations of NEM (0.1–1mM) drastically reduced H+-ATPase activity. The inhibiting effect of PM treatment with NEM was stronger when the H+-ATPase activity was assayed at pH values (7.1–7.2) higher than that optimal for enzyme activity (6.3). If the PM H+-ATPase was activated by proteolytic cleavage of the C-terminal domain or by its displacement by fusicoccin prior to NEM treatment, the inhibitory effect of NEM on the W-ATPase activity became independent of the pH of the assay medium. Moreover, inhibition by NEM of H+-ATPase activity also became independent of the pH of the assay medium if the C-terminal was proteolytically cleaved or displaced by lysophosphatidylcholine after NEM treatment of the PM. Controlled trypsin treatment of NEM-treated PM produced, beside the 90 kDa truncated PM H+-ATPase, fragments of 60 to 30 kDa of the enzyme that were undetectable after trypsin treatment of control PM. These results indicate that PM treatment with NEM modifies the H+-ATPase conformation, exposing trypsin cleavage sites scarcely accessible in control PM and strengthening the autoinhibitory action of the C-terminal domain.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.