植物质膜H+-ATPase的研究进展

质膜H -ATPase参与植物细胞的物质跨膜转运、细胞的伸长生长、气孔的开闭以及植物对环境胁迫的响应等生理过程,是植物生命活动的“主宰酶”。其活性调节涉及激素、环境因子等多种因素,可发生在转录、翻译和酶分子等多级水平。因此,在植物生长发育过程中,质膜H -ATPase活性的调节对生理活动起重要作用。本文就植物质膜H -ATPase的结构特征、生理功能、活性变化及其调节机理等的研究进展进行综述,以进一步揭示该酶的生理功能及其调节机理与植物生命活动过程的关系。     

植物质膜H+-ATPase响应盐胁迫的分子机制

植物细胞质膜质子泵（PM H^＋-ATPase）有看家酶之称,是由多基因编码的,其主要功能是向细胞营养物质的吸收和离子跨膜运输提供驱动力。文章介绍PM H^＋-ATPase在植物抗盐中的作用及研究进展。     

植物质膜水通道蛋白转运及逆境胁迫响应的分子调控机制

质膜水通道蛋白即质膜内在蛋白(plasma membrane intrinsic proteins, PIPs),属于通道蛋白,定位在质膜上,是植物体内水分子、CO₂及其他一些小分子溶质跨细胞膜运输的通道。PIPs对运输基质具有高度选择性,在维持植物细胞的水分平衡过程中发挥重要作用。PIPs的表达、活性与定位不但受转录水平和翻译后水平的调控,而且受外界环境影响。研究表明在非生物胁迫下,PIPs表达模式和定位会发生改变。本文重点阐述了PIPs转运的分子机制、转录水平及翻译后水平的调控机制以及PIPs对非生物胁迫的响应机制,分析了目前关于PIPs的研究动态和值得探究的研究方向,以期帮助相关领域的科研人员对PIPs的研究进展有更深入地了解。     

拟南芥液泡膜Na+/H+逆向转运蛋白的研究进展

拟南芥液泡膜Na /H 逆向转运蛋白是由AtNHX1基因编码的一个在盐胁迫中起重要作用的蛋白。本文综述了AtNHX1的基本结构、功能及作用机制,展望其作为有效植物耐盐基因的前景,并对拟南芥液泡膜Na /H 逆向转运蛋白基因家族其他成员的研究,也做了相应的概括。     

壳梭孢素对质膜H+-ATPase活力影响机制的研究进展

壳梭孢素（FC）作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统,FC刺激质膜H+_ATPase的活性是通过FC结合蛋白（FCBP）与H+_ATPase 发生作用。FCBP是14-3-3蛋白家族成员之一。     

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。     

Cu和丹皮酚磺酸钠处理对凤丹根系生长、丹皮酚含量及H+-ATPase活性的影响

采用水培法,研究了Cu单一处理及Cu与EGTA和丹皮酚磺酸钠(SPS)复合处理对凤丹(Paeonia suffruticosa ‘Feng Dan’)幼苗根长、根系中Cu和丹皮酚含量的影响,并研究了Cu和SPS单一及复合处理对幼苗根系离体质膜和液泡膜微囊H+-ATPase活性的影响.结果显示:经5μmol·L-1Cu处理后凤丹幼苗根长和丹皮酚含量均略高于对照但总体上差异不显著;经10、20和30 μmol·L-1 Cu处理后,幼苗根长和丹皮酚含量总体上均低于对照,且随Cu浓度增加和处理时间延长,降幅增大.与10 μmol·L-1Cu单一处理相比,10 μmol·L-1Cu- 10μmol·L-1EGTA和10 μmo1.L-1Cu-10 μmol·L-1 SPS复合处理均可以使根系中Cu含量显著降低、丹皮酚含量显著提高;其中,10μmol·L-1Cu-10 μmol·L-1EGTA处理组幼苗根系中Cu含量的降低幅度最大,而10 μmol·L-1 Cu-10 μmol·L-1SPS处理组幼苗根系中丹皮酚含量的增加幅度最大且显著高于对照.与对照相比,经5、10和20μmol · L-1 Cu单一处理后丹凤根系离体质膜和液泡膜微囊H+-ATPase活性均降低,且随Cu浓度提高降低幅度增大;而经0.1、0.2、0.5和1.0μmol·L-1SPS单一处理总体上可使膜微囊H+-ATPase活性逐渐增加;与10 μmol·L-1Cu单一处理相比,10 μmol·L-1Cu与0.1、0.2和0.5 μmol·L-1SPS复合处理均可使膜微囊H+- ATPase活性提高,且H+-ATPase活性均呈现随SPS浓度提高逐渐增加的趋势.研究结果揭示:较高浓度Cu胁迫对凤丹幼苗根系生长及丹皮酚合成以及质膜和液泡膜微囊H+-ATPase活性均有明显抑制作用,但添加外源丹皮酚磺酸钠对Cu胁迫伤害具有一定的缓解效应.     

细胞内物质转运调节分子ARFGAP3的克隆表达及生化活性

ADP核糖基化因子-GTP酶活化蛋白(ARF GAP)是重要的细胞内物质转运调节分子.在22周孕龄人胎肝cDNA文库中发现一种新基因,其编码的氨基酸序列与大鼠ARF1 GAP有32%同源性.将这种新基因命名为“ARFGAP3”,对其进行功能研究,利用逆转录-聚合酶链式反应(RT-PCR),从人胎盘总RNA中扩增ARFGAP3全长cDNA序列,并将其亚克隆到pGEM-T载体;采用RNA印迹法和斑点杂交法,检测其组织表达谱,发现在多种腺体和睾丸中有很高水平ARFGAP3基因转录,并且只有一种约2.7 kb的转录本.利用基因重组技术,构建表达质粒pBAD/Thio-ARFGAP3,在大肠杆菌中表达,采用亲和层析法纯化表达产物,利用肠激酶切除重组融合蛋白N端引导序列.检测重组ARFGAP3的生化活性,证实ARFGAP3对ARF1具有GAP活性,促进ARF1结合的GTP水解为GDP,磷脂酰肌醇二磷酸(PIP2)增强其GAP活性,而磷脂酰胆碱(PC)抑制其GAP活性.     

豚鼠精子质膜Ca2+ -ATPase活性与精子顶体反应关系的研究

用生化测定法首次证实豚鼠精子质膜Ca²⁺-ATPase活性在精子获能和顶体反应过程中显著下降.Ca²⁺-ATPase抑制剂利尿酸(ethacrynic acid)抑制质膜Ca²⁺-ATPase活性,但钙调素(50μg/mL)的拮抗剂三氟拉嗪(TFP,200～500μmol/L)对该酶活性没有影响,说明钙调素不直接参与精子依赖于ATP的Ca²⁺的主动泵出.但钙调素与精子的Ca²⁺内流有关,钙调素拮抗剂TFP显著促进精子顶体反应和精子对Ca²⁺的摄入.Ca²⁺-ATPase抑制剂栎皮酮(quercetin)、原钒酸钠(sodiumorthovandate)、利尿磺胺(furosemide)和利尿酸均显著促进豚鼠精子的顶体反应,但却抑制精子对Ca²⁺的摄入,这无法用它们对质膜Ca²⁺-ATPase活性的抑制作用解释.推测这可能是由于Ca²⁺-ATPase抑制剂在抑制质膜Ca²⁺-ATPase活性的同时也抑制了顶体外膜或线粒体外膜上的该酶的活性,导致Ca²⁺在细胞质内的积累,进而通过负反馈机制抑制Ca²⁺进一步内流所致.另外,Ca²⁺-ATPase抑制剂对糖酵解的抑制作用也可能是Ca²⁺在细胞质中积累和抑制精子Ca²⁺摄入的原因.     

质膜H+-ATPase与环境胁迫

植物根系质膜H -ATPase在调节细胞内pH值,促进养分吸收、同化物运输等方面具有重要作用。对质膜H -ATPase的结构、功能和分子机制进行综述,并讨论了质膜H -ATPase在信号传递过程及植物适应环境胁迫中的作用,最后就植物质膜H -ATPase的研究及应用提出几点看法。     

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

Abstract: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H⁺-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H⁺-ATPase activity both in PM isolated from germinating radish ( Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H⁺-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H⁺-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H⁺-ATPase and 14-3-3 protein.     

The Ca2+ Channel TRPML3 Regulates Membrane Trafficking and Autophagy

TRPML3 is an inward rectifying Ca²⁺ channel that is regulated by extracytosolic H⁺. Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca²⁺ in the vicinity of cellular organelles that is necessary to regulate these cellular events.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

酸胁迫对干酪乳杆菌H+-ATP酶基因表达的影响

耐酸性是乳酸菌重要的益生菌性状之一。实验采用半定量RT-PCR法分别对在不同酸度条件下培养后的3株不同干酪乳杆菌的H -ATP酶基因mRNA表达水平进行了比较和分析。实验结果显示,随着培养基酸度增加干酪乳杆菌的生长受到抑制,特别在pH4.0条件下干酪乳杆菌的生长受到强烈的抑制;H -ATP酶基因的表达量随着培养基酸度的增加而增加。推测H -ATP酶与干酪乳杆菌耐受酸性条件是有一定的关联的。     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Modulation of the Ca2+,Mg2+-ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Modifications of Synaptosomal Na+-K+-ATPase Activity During Vasogenic Brain Edema in the Rabbit

This study investigates the functioning of synaptosomal ouabain-sensitive Na+ -K+ -ATPase in cold-induced edema. During vasogenic brain edema development, the enzyme affinities for Na+ and K+ are progressively decreased paralleling the increase in the tissue water content, whereas maximal velocity of the reaction is not changed. On the basis of these data, it is likely that Na+ -K+ -ATPase impairment accounts for the intracellular uptake of water in this model of edema.