Ca^(2+)-ATPase参与植物耐盐性调控的研究进展

盐胁迫下细胞质Ca^(2+)浓度升高,细胞会激活Ca^(2+)调节的靶酶或者与Ca^(2+)高度亲和的受体蛋白,其中,与Ca^(2+)高度亲和的受体蛋白中,植物钙泵(Ca^(2+)-ATPase)是P型ATP酶,包含内质网Ca^(2+)-ATPases与质膜Ca^(2+)-ATPas‐es,通过主动运输将Ca^(2+)从细胞质转移到质外体或细胞器。大量研究表明,植物的耐盐性在很大程度上与其维持钙泵即Ca^(2+)-ATPase活性的能力有关。多种植物Ca^(2+)-ATPase对盐胁迫表现出敏感性,并受到外源Ca^(2+)的保护,表明外源钙处理与Ca^(2+)-ATPase活性可能在盐胁迫下的细胞内钙稳态和信号转导中起重要作用。该研究概述了植物Ca^(2+)-ATPase类型、结构与性质,亚细胞定位Ca^(2+)-ATPase及外源钙与亚细胞定位Ca^(2+)-ATPase参与植物耐盐调控研究进展,重点对质膜、液泡膜、核膜、内质网及高尔基体Ca^(2+)-ATPases参与植物耐盐调控的研究进展进行了综述,并提出展望。该研究为了解植物耐盐性生理及分子机制提供帮助,同时为作物耐盐栽培提供新思路。

Contribution of plasma membrane Ca^(2+) ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca2+ATPase,isoform 2 in the mammalian brain.This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex;i.e. the Purkinje neurons(PNs) .Here we review recent evidence,including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2(PMCA2) knockout mouse,to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour.These studies have also revealed that deletionof PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development,they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

Role of platelet plasma membrane Ca^(2+)-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

The investigation for the relationship among serum leptin, erythrocyte membrane Ca^(2+)-ATPase activity and hypertensive disorder complicating pregnancy

Objective： To study the significance of Leptin and the activity of erythrocyte membrane Ca^2＋-ATPase（EMCA） in the development of hypertensive disorder complicating pregnancy. Methods： Radioimmunoassay was used to test the level of serum Leptin, and the activity of EMCA was determined chemically in 38 pregnant women with hypertensive disorder complicating pregnancy and 36 normotensive pregnant women. Results： The level of serum Leptin in hypertensive disorder complicating pregnancy（gestational hypertension： 13.76 ± 3.46 ng/ml; preeclampsia： 15.76 ± 5.47 ng/ml; eclampsia： 18.32 ± 6.38 ng/ml）was significantly higher than that in normotensive pregnant women （11.33 ± 2.93 ng/ml） ,respectively. The average EMCA activity of patients with hypertensive disorder complicating pregnancy （gestational hypertension： 1.65 ± 0.24 μmol·pi/mg.h ; preeclampsia： 1.37 ± 0.19 μ mol·pi/mg·h;eclampsia：1.12 ± 0.14 μmol·pi/mg·h） was significantly lower than that of normotensive pregnant women（1.83 ± 0.38 μ mol·pi/mg·h）,respectively. There was a negative correlation between the level of serum Leptin and the activity of RMCA in hypertensive disorder complicating pregnancy （r = -0.63）. Conclusion： Inhibition of EMCA activity of erythrocyte in hypertensive disorder complicating pregnancy may increase cytoplasmic free calcium, which contributes to the development of hypertensive disorder complicating pregnancy. The negative correlation between the level of serum Leptin and the activity of EMCA, also suggested that serum Leptin and the activity of EMCA may play a role in the development of hypertensive disorder complicating pregnancy.

质膜Ca^2＋-ATPase的结构与功能

质膜Ca2+-ATPase(PMCA)是P型ATPase家族的一员,在真核细胞中主要负责信号刺激后胞内高浓度Ca2+的清除扫尾工作,并对维持静息状态下较低Ca2+浓度起着重要的调节作用.PMCA的一级结构已被确定,拓扑学结构显示,它有10个跨膜区和3个胞浆功能区.它的4个编码基因可产生4种亚型(PMCA 1~4),这些亚型在功能与分布上存在差异.PMCA的活性可被钙调蛋白等多种因素调节,这与其结构特征息息相关.近年来,PMCA已被证实与脂筏结构有一定关联,它在信号传导和细胞凋亡中的作用也成为目前科学研究的焦点.本文主要对PMCA的结构、亚型和功能的研究现状进行综述.

大白菜干烧心病发生过程中Ca^(2+)-ATPase活性的变化

人为诱导大白菜干烧心病发生，通过超速离心制备心叶组织细胞膜微囊，测定各细胞器膜的Ｃａ２＋－ＡＴＰａｓｅ活性变化。结果表明，随缺钙天数增加，细胞膜总Ｃａ２＋－ＡＴＰａｓｅ、ＰＭ－Ｃａ２＋－ＡＴＰａｓｅ和ＴＮ－Ｃａ２＋－ＡＴＰａｓｅ活性迅速上升，在处理后第３天达到高峰，随后逐渐下降，而潜在性Ｃａ２＋－ＡＴＰａｓｅ活性却呈逐渐上升趋势。同时，细胞膜透性和丙二醛含量逐渐增大，超氧化物歧化酶（ＳＯＤ）和过氧化物酶（ＰＯＤ）活性则逐渐下降。推测该病的发生与细胞膜钙泵活性变化诱导的钙的第二信使功能有关。

草莓采后微粒体膜Ca^(2+)-ATPase活性与膜脂过氧化水平

测定了常温 (2 5℃ )和低温 (4℃ )贮藏的草莓果实中微粒体膜Ca2 + ATP酶 (Ca2 + ATPase)与超氧化物歧化酶 (SOD)活性、超氧阴离子 (O-·2 )产生速率和丙二醛 (MDA)含量的变化。结果显示 ,随果实衰老 ,低温贮藏的果实微粒体膜Ca2 + ATPase总活性和质膜、线粒体膜及液泡膜上的Ca2 + ATPase活性 ,微粒体膜SOD活性和O-·2 产生速率呈先升高后降低的趋势 ,MDA含量变化与之相反 ;常温贮藏的果实Ca2 + ATPase活性和O-·2 产生速率变化较为迅速 ,与低温类似 ,而SOD活性逐步下降 ,MDA含量不断升高。这些表明 ,草莓果实衰老与细胞质内Ca2 +

外源乙烯对桃果实微粒体膜Ca^(2+)-ATPase活性和膜脂过氧化水平的影响

以'迎庆'桃果实为材料,研究了经外源乙烯(50μL/L)处理12h后,在常温(25℃)下贮藏过程中,果实微粒体膜Ca2+-ATPase活性和膜脂过氧化水平的变化。结果表明:外源乙烯促进果实内源乙烯的生成,刺激膜Ca2+-ATPase活性先上升而后下降,同时加速超氧自由基的产生,提高膜脂过氧化产物丙二醛含量,增强了膜脂过氧化作用;钙调素拮抗剂三氟拉嗪(TFP)和钙通道阻塞剂异博定(VER)均在一定程度上抑制乙烯诱导的上述效应,这表明细胞内Ca2+和CaM参与了外源乙烯反应。

桃果实采后微粒体膜Ca^(2+)-ATPase活性与膜脂过氧化

以常温(25℃)和低温(4℃)贮藏的迎庆桃果实为试验材料,对其果实硬度、呼吸强度进行了测定,并对微粒体膜Ca2+-ATPase、超氧化物歧化酶(SOD)活性、氧自由基变化和膜的伤害程度进行了研究.结果表明,随桃果实衰老,常温贮藏的果实硬度迅速下降、微粒体膜上的Ca2+-ATPase活性、SOD活性和O2-产生速率均呈跃变式变化,先升高后降低;膜脂过氧化产物MDA的含量逐渐增加;与常温相比,低温可以抑制果实硬度的下降、呼吸速率、Ca2+-ATPase和SOD活性的下降及推迟峰值的出现,同时降低O2?-产生速率和MDA含量.以上结果表明,桃果实衰老与细胞质内Ca2+稳态的破坏和膜脂过氧化作用的加强有密切关系.

渗透胁迫下外源试剂对玉米幼苗微粒体膜Ca^2＋-ATPase活性的影响

以钙离子鳌合剂EGTA、钙通道阻断剂Vp、钙调素拮抗剂TFP和ABA分别处理玉米幼苗,对渗透胁迫下根系和叶片质膜、液泡膜、线粒体膜以及总微膜Ca2+-ATPase活性进行了测定。结果表明,正常条件下,叶片和根系微膜系统总Ca2+-ATPase活性随光照时间延长出现一个峰值。总体比较,渗透胁迫下三种膜上Ca2+-ATPase活性相当,而且均随渗透胁迫发生不同程度的变化。就微膜系统总酶活变化而言,叶片高于根系,渗透胁迫降低叶片酶活;而渗透胁迫对根系酶活性影响不大。分解到三种膜上,渗透胁迫对叶片酶活的提高均表现为正效应,根系上,对线粒体膜和液泡膜酶活提高表现为正效应,而对质膜酶活表现为显著抑制。外源试剂处理发现,ABA和EGTA在叶片上均可逆转PEG降低总酶活的效应,且在处理6 h时使得酶活性显著超过正常水平;而根系上表现不一:其中,Vp对质膜上PEG提高酶活效应抑制作用较大,且显著提高线粒体膜酶活;ABA显著提高液泡膜上酶活性;TFP处理在胁迫前期基本表现为促进作用,而6 h之后大都表现为抑制效应。说明:渗透胁迫下,玉米叶片质膜、液泡膜以及线粒体膜均参与了胞质钙离子稳态的调控过程,外源试剂处理对Ca2+-ATPase具不同程度的调节作用,叶片和根系Ca2+-ATPase对外源试剂响应存在差异性。

人晶状体上皮细胞系细胞膜Ca^(2+)-ATPase同工异构体2的表达

目的 检测人晶状体上皮细胞系B 3(HLEB 3)中,细胞膜钙ATP酶(PMCA)的同工异构体之一,PMCA2的表达情况。 方法 细胞培养HLEB 3,当细胞培养达 90%融合时,提取其总mRNA,用PMCA2特异性引物,用反向聚合酶链反应(RT PCR)法检测PMCA2在mRNA水平上的表达。提取细胞膜蛋白,经SDS PAGE电泳以及蛋白免疫印记法(Westernblot),用抗 PMCA2的特异抗体检测其在蛋白质水平上的表达。 结果 RT PCR显示PMCA2特异性引物在HLEB 3细胞扩增出唯一 550bp的片段。蛋白免疫印记法证实PMCA2的表达,相对分子质量约为126 000。 结论 HLEB 3细胞中,在mRNA水平以及蛋白质水平均有PMCA2的表达。并有可能在HLEB 3细胞的钙离子平衡调节中发挥重要作用。

烟草叶和根细胞质膜Ca^(2+)-ATPase特性的比较研究

测定了烟草叶和根细胞质膜Ca2+-ATPase的活性并对它们的特性进行了比较.结果表明,二者在许多方面都不尽相同.前者的最适pH为7.0,后者则为6.0,而且在一个相对较宽的pH范围内有高的活性.叶细胞质膜Ca2+-ATPase的最适反应温度为45℃,[ATP]0.5为2.2mmol/L,根细胞质膜Ca2+-ATPase则分别为40℃和1.9mmol/L.叶和根的细胞质膜Ca2+-ATPase均受Ca2+激活.曙红B(EB)对烟草叶和根细胞质膜Ca2+-ATPase有明显的抑制作用,当EB浓度达到50μmol/L后,抑制作用才不再有显著的增加.本文讨论了烟草叶和根细胞质膜Ca2+-ATPase特性出现差异的原因及其与环境的关系.

GM1对肌质网Ca^(2+)-ATPase活性及膜流动性的影响

外源性GM1对肌质网Ca2+-ATPase的水解及转运活性都有明显的抑制作用.在GM1浓度为0～8nmol/mg蛋白质范围内抑制作用具有浓度依赖性.当GM1浓度达到8nmol/mg蛋白质时,酶活性受到最大抑制,此时水解活性降低51%,转运活性降低49%.荧光偏振测定结果表明:GM1参入后,肌质网膜流动性降低.

腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca^(2+)-ATPase活性和[Ca^(2+)]i的作用

目的:探讨腺相关病毒载体介导的磷酸受纳蛋白(PLB)反义RNA对肌浆网Ca2+-ATPase活性以及细胞内钙浓度([Ca2+]i)的作用。方法:构建PLB反义RNA的重组腺相关病毒载体(rAAV-asPLB)和携带报告基因LacZ的重组腺相关病毒载体(rAAV-LacZ)。分别转染培养的大鼠心肌细胞,测定PLBmRNA和蛋白质表达,检测肌浆网Ca2+-ATPase活性和[Ca2+]i。结果:RT-PCR和Westernblotting显示,感染rAAV-asPLB的心肌细胞的PLBmRNA和蛋白质表达低于正常对照和感染rAAV-LacZ组;而Ca2+-ATPase活性大于正常对照和感染rAAV-LacZ组。激光共聚焦显微镜检测显示,静息状态时,rAAV-asPLB感染组[Ca2+]i降低;异丙肾上腺素刺激后,各组[Ca2+]i均升高,但是rAAV-asPLB感染组[Ca2+]i变化幅度大。结论:腺相关病毒介导的PLB反义RNA对大鼠心肌细胞PLB表达具有抑制作用,增强Ca2+-ATPase活性,降低静息状态的[Ca2+]i,增加异丙肾上腺素刺激后[Ca2+]i的变化幅度。

糖尿病视网膜病变患者红细胞膜Ca^(2+)-Mg^(2+)-ATPase活性变化及相关研究

目的探讨糖尿病视网膜病变发生发展的有关因素。对40例糖尿病机网膜病变患者红细胞膜Ca^(2+)－Mg^(2+)－ATPasc活性进行了检测，并与红细胞内Ca^(2+)－Ms^(2+)－ATPasc含量、空腹血糖、糖基化血红蛋白及红细胞变形指数等进行了分析。结果糖尿病机网膜病变患者红细胞膜Ca^(2+)－Ms^(2+)－ATPasc活性显著下降（P＜0．01），细胞内Ca^(2+)含量增加（P＜0．01）而Mg^(2+)含量则显著下降（P＜0．01）。糖尿病视网膜病变者红细胞膜Ca^(2+)－Ms^(2+)－ATPasc活性下降与空腹血糖、糖基化血红蛋白、红细胞变形指数及红细胞内Ca^(2+)－Ms^(2+)－ATPasc含量等变化密切相关。结论糖尿病视网膜病变患者红细胞膜Ca^(2+)－Ms^(2+)－ATPasc活性异常下降可能是导致或加速视网膜病变发生发展的原因之一。

卒中型高血压大鼠心肌线粒体Ca^（2＋），Mg^（2＋）—ATPase及膜流动

本文比较了卒中型自发往高血压鼠（ＳＨＲｓｐ）和京都Ｗｉｓｔａｒ正常血压鼠（ＷＫＹ）心肌线粒体Ｃａ￣（２＋），Ｍｇ￣（２＋）－ＡＴＰａｓｅ活力及膜流动性。用定磷法测得ＷＫＹ和ＳＨＲｓｐ的Ｃａ￣（２＋），Ｍｇ￣（２＋）－ＡＴＰａｓｅ活力（２５℃）分别为０．２８７±０．０１６及０．２１８±０．０１７μｍｏｌ／（ｍｉｎ．ｍｇ）（－ｘ±Ｓｘ）。ＳＨＲｓｐ心肌线粒体Ｃａ￣（２＋），Ｍｇ￣（２＋）－ＡＴＰａｓｅ活力降低２４．７％，两组比较有显著差异（Ｐ＜０．０１，ｎ＝９）．以ＤＰＨ标记心肌线粒体膜，测得ＷＫＹ和ＳＨＲｓｐ的荧光偏振值分别为０．１８７±０．００３及０．１８１±０．００３（Ｐ＞０．０５，ｎ＝６）。

Changes in erythrocyte membrane ATPases and plasma lipid peroxides in upper abdominal surgery under intravenous procaine-balanced anesthesia

AIM To observe the changes in erythrocyte membrane ATPases and plasma lipid peroxides (LPO) patients with in abdominal surgery under intravenous procaine-balanced anesthesia.METHODS By determining the ATPase activities of erythrocyte membrane, effects of upper abdominal surgery under intravenous procaine-balanced anesthesia on the function of erythrocytes were observed in 15 patients undergoing cholecystectomy and gastrectomy (5 males and 10 females, aged 45.9±10.20 years and weighed 60.60kg±11.93kg). All patients were free from severe renal, hepatic, pulmonary, cardiac, metabolic and endocrinological diseases and acute infection for at least 2 weeks before surgery. Patients receiving any drug known to affect carbohydrate metabolism prior to anesthesia were excluded from the study.RESULTS Erythrocyte membrane Na+, K+-ATPase, Mg2+-ATPase, Ca2+, Mg2+-ATPase activities were not significantly changed 60min-90min after incision as compared with 30min before anesthesia, but were decreased markedly 10min and 24 hours after completion of operation (P＜0.01). Plasma lipid peroxides (LPO) were increased significantly 24 hours after surgery (P＜0.01) following an initially marked but transient reduction. Plasma LPO changes were not correlated with erythrocyte membrane ATPase activities, r=-0.0396, -0.0097 and 0.4383, respectively (P＞0.05).CONCLUSION Abdominal surgical trauma under intravenous procaine-balanced anesthesia may be associated with the decreased ATPase activities of erythrocyte membrane and increased LPO in plasma.

质膜Ca^(2+)-ATPase和钙调节异常在恶性肿瘤发生发展中的作用及研究进展

钙离子(Ca^(2+))作为细胞内普遍存在的第二信使在细胞的许多生理病理过程中发挥了重要作用。而质膜Ca^(2+)-ATPase(PMCA)是一种依赖ATP的驱动将细胞内的Ca^(2+)排出胞外的钙泵。它的表达异常将会导致细胞内Ca^(2+)平衡的紊乱以及各种疾病的发生。由于Ca^(2+)和神经传递、肌肉收缩密切相关,早期的研究大多集中在中枢神经系统、心血管系统。近年来,随着研究的进展。很多学者逐渐发现钙信号在一些恶性肿瘤的进程中发挥了重要作用,比如增殖、迁移等。很多研究也发现了在各种恶性肿瘤中PMCA表达的改变,包括乳腺癌,结直肠癌、胰腺癌等。同时,在一些恶性肿瘤细胞系中发现并证实了PMCA各种亚型的存在及其差异性的表达。一些研究者还在一些临床肿瘤标本中检测了PMCA各种亚型的表达水平,而且对其表达水平改变的机制进行了初步的研究并提出在某些特异性的恶性肿瘤中PMCA有可能成为潜在的治疗靶点。因此,该文将对PMCA和钙调节异常在各种恶性肿瘤发生发展中的作用及研究进展进行综述。

质粒DNA对大鼠骨骼肌肌质网Ca^(2+)-ATPase活性及ryanodine受体结合反应的影响

目的：观察质粒ＤＮＡ 对离体大鼠骨骼肌肌质网Ｃａ２＋ＡＴＰａｓｅ 活性及ｒｙａｎｏｄｉｎｅ 受体结合反应的影响。方法：参照Ｊｏｎｅｓ 等的方法比色测定肌质网Ｃａ２＋ＡＴＰａｓｅ 活性；以３Ｈｒｙａｎｏｄｉｎｅ 作为配基，液闪测定肌质网Ｃａ２＋ 释放通道ｒｙａｎｏｄｉｎｅ 受体与配体的结合。质粒ＤＮＡ 处理组预先将肌质网蛋白与质粒ＤＮＡ３７ ℃共同孵育３０ ｍｉｎ ，而后再测定肌质网Ｃａ２ ＋ＡＴＰａｓｅ 活性和ｒｙａｎｏｄｉｎｅ 结合反应。结果：预先用１０、２０ 和３０ μｇ ｐｃＤＮＡ３ 及ｐｃＤＮＡ３／ＡＮＦ处理后，肌质网Ｃａ２＋ＡＴＰａｓｅ 活性分别较对照组升高４５％ （Ｐ＜ ０．０５） ，６８ ％（ Ｐ＜ ０．０１） 和１０９ ％ （ Ｐ＜ ０．０１） 及１０９ ％ ，１１８％ 和１４５％ （Ｐ值均小于０ ．０１） ；质粒ｐｃＤＮＡ３ 可明显降低ｒｙａｎｏｄｉｎｅ 受体的亲和力，并使Ｂｍａｘ 增强。结论：质粒ＤＮＡ可能通过增强肌质网Ｃａ２ ＋ＡＴＰａｓｅ 的活性促进肌质网对Ｃａ２ ＋ 的摄入，通过影响ｒｙａｎｏｄｉｎｅ 受体而调控肌质网Ｃａ２ ＋ 释放。