CaMBP—10介导的质膜H^＋—ATP酶磷酸化对该酶活性的调节

ＣａＭＢＰ－１０在活体处理条件下,抑制ＩＡＡ诱导的质膜Ｈ＾＋－ＡＴＰ酶活性及其磷酸化,抑制作用可被ＩＡＡ逆转并在外加ＣａＭ时被消除,与前期ＢＰ－１０对ＩＡＡ生理应答的调节效应相吻合。并且在各项处理中,质膜Ｈ＾＋－ＡＴＰ酶活性与其磷酸化水平呈现极显著的正相关。     

一种钙调素结合蛋白对小麦质膜H~ -ATP酶活性的调节

植物生长素参与植物生长和发育诸多方面的调节。研究表明,生长素的调节机制与Caz”的存在紧密相关。Caz”在植物激素的信号传导中起着信使作用（Hepler和Randy1985）,它与钙调素（Ca．－－－urr－ulin,CaM）结合参与了各种类型植物激素应答反应的调节。现已查明,CaM的诸多功能常受其内源性结合蛋白的调控。本研究组曾分离得到一种新的植物CaM结合蛋白——CaMBP－10o实验证明,CaMBP－10通过与CaM的特异性结合显著抑制了CaM对其靶酶的激活（尚克进等1991）。前期工作还发现CaMBP－10对生长素诱导的小麦芽鞘伸长和质子外排均有…     

转脂蛋白CaMBP10的胶体金标记及对白菜中CaMBP10结合蛋白的鉴定

植物转脂蛋白 (LTP)是一类广泛存在于高等植物中的空间结构高度保守的碱性小分子蛋白,其确切功能和调节机制至今仍不清楚.本室从白菜中分离的钙调素结合 蛋白10 (CaMBP10),经序列分析被鉴定为植物转脂蛋白家族成员.近期研究结果表明 ,CaMBP10 参与了植物的生物与非生物胁迫反应.为了深入探讨CaMBP10的抗性机制,确定植物中与其相互作用的蛋白质,本文拟建立胶体金标记CaMBP10 的方法,通过凝胶覆盖分析,检测植物样品中的CaMBP10 结合蛋白为此,对标记反应的最适条件进行了优化,确定最佳条件为：交联剂戊二醛用量为0.034%,交联反应pH值为7 .0,交联反应时间为40 min,胶体金颗粒度为10 nm,胶体金溶液的pH为7.0. 本文确定建立了植物样品中CaMBP10结合蛋白的分析与鉴定方法.     

植物钙调素结合蛋白研究进展

钙调素(CaM)作为最重要的一类Ca2 传感蛋白可以通过与其下游CaM结合蛋白(CaMBP)作用而调节细胞的生理功能.因此,对CaMBP的研究是揭示CaM作用机制的重要内容,是探明Ca2 -CaM信号转导系统的关键.该文从CaMBP和CaM的结合特性、植物CaMBP的分布以及植物CaMBP的生物学功能等方面综述了植物CaMBP的研究现状和最新进展.     

白菜转脂蛋白CaMBP10分子中钙调素结合结构域的鉴定

植物转脂蛋白(LTPs)是多基因编码的蛋白家族, 广泛分布于高等植物,其确切的生理功能至今仍不清楚. 本室从白菜中分离的钙调素结合蛋白-10 (CaMBP10) 经序列分析 被鉴定为植物转脂蛋白家族成员,体外实验证明钙调素(CaM)调节其脂质结合活性.为了深入了解转脂蛋白与CaM的相互作用机制,本文通过删除、缺失和定点突变等分子生物学手段确定了白菜转脂蛋白CaMBP10分子中的钙调素结合结构域.该结构域位于分子C末端 64~83位氨基酸残基之间,其中疏水氨基酸的分布具有1-5-8-10 的CaM结合模序特征.     

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。     

豌豆质膜内源CDPK对植物转脂蛋白CaMBP-10的磷酸化及CaMBP-10对激酶自磷酸化的影响

植物转脂蛋白（LTPs）是多基因编码的蛋白家族,广泛分布于高等植物.虽然LTPs的确切功能至今仍不完全清楚,但它参与植物生物、非生物胁迫反应以及它的抗性功能已成为近年来的研究热点.关于LTPs功能的调节机制目前几乎一无所知.最近,从白菜中分离的钙调素结合蛋白-10（CaMBP-10）被鉴定为植物转脂蛋白家族成员,并且,体外实验证明钙调素（CaM）调节其脂质结合活性.为了深入了解转脂蛋白功能的调节机制,本文研究了CaMBP-10的磷酸化作用,发现CaMBP-10可被豌豆质膜内源性蛋白激酶磷酸化,钙离子（Ca2＋）能刺激磷酸化,钙螯合剂EGTA以及CaM拮抗剂W-7和TFP均能显著抑制磷酸化.免疫印迹分析最终确定该激酶为CDPK家族成员.构建突变体进一步研究了CaMBP-10的磷酸化位点,发现其位于蛋白的C-末端区域,并与已确定的CaM结合位点重合.同时,分析结果表明CaM能抑制CaMBP-10的磷酸化.反之,CaMBP-10的磷酸化又能阻断其与CaM的结合,显示出两种调节方式相互竞争的特点.为深入研究磷酸化作用对CaMBP-10脂质结合活性的影响,构建突变体（Ser83Asp,Ser85Asp）以模拟磷酸化状态.实验结果显示,磷酸化作用能显著增强CaMBP-10的脂质结合活性,而且突变体的脂质结合活性不受CaM的影响.采用胶内磷酸化测定法（in-gelkinaseassay）研究了激酶的自磷酸化特点以及CaMBP-10对激酶自磷酸化的影响,发现CaMBP-10能激活激酶的自磷酸化,激酶的自磷酸化又能促进其对底物的磷酸化作用.这样,激酶的自磷酸化与底物的磷酸化形成一种＂正反馈环＂的调节模式.综合研究结果,本文首次证明了LTP受CaM结合和CDPK磷酸化的双重调节.而且,CaM结合位点与磷酸化位点的重合预示可能存在特殊的调节机制,以协同应答胞内的Ca2＋信号.     

IAA对小麦胚芽鞘质膜蛋白磷酸化的影响

磷酸化／脱磷酸化机制是众多信号过程中的重要环节,很多信号物质被细胞受体识别后引发蛋白激酶和蛋白磷酸酶活性变化,通过磷酸化／脱磷酸化进一步调节多种酶活性而产生各种生理效应。在对生长素ＩＡＡ的信号转导的研究中,发现ＩＡＡ处理的小麦胚芽鞘质膜蛋白中蛋白激酶的活性和蛋白磷酸化程度都发生改变,并找到两种受到调节的蛋白激酶。钙离子通道抑制剂ＬａＣｌ３阻断了ＩＡＡ的这种作用,表明Ｃａ％２＋参与了ＩＡＡ的信号转导     

IAA对小麦胚芽鞘质膜蛋白磷酸化的影响

磷酸化／ 脱磷酸化机制是众多信号转导过程中的重要环节,很多信号物质被细胞受体识别后引发蛋白激酶和蛋白磷酸酶活性变化,通过磷酸化／ 脱磷酸化进一步调节多种酶活性而产生各种生理效应。在对生长素ＩＡＡ 的信号转导的研究中,发现ＩＡＡ 处理的小麦胚芽鞘质膜蛋白中蛋白激酶的活性和蛋白磷酸化程度都发生改变,并找到两种受到调节的蛋白激酶。钙离子通道抑制剂ＬａＣｌ３ 阻断了ＩＡＡ 的这种作用,表明Ｃａ２＋参与了ＩＡＡ的信号转导过程。     

CaMBP-10单克隆抗体的制备及CaMBP-10在豌豆器官中的表达检测

用电泳纯钙调素结合蛋白BP 10 (CaMBP 10 )免疫小鼠 ,制备单克隆抗体 (McAb) .用MEP(mercapto ethtyl pyridine)HyperCel疏水层析柱从细胞培养上清中纯化并获得单克隆抗体 ,同时测定了抗体 抗原反应的基本特性 .此单克隆抗体具有较高纯度、特异性和亲和力 .亲和常数 (Kaff)为1 2 6× 10 9(mol L) -1,此抗体和CaM在空间上以相同或相近的位点与CaMBP 10相结合 .以胶体金标记的抗体为探针 ,研究CaMBP 10在豌豆幼叶、成熟叶、茎尖、茎、根等不同器官的分布特征 ,并与胶体金标记CaM的结合情况相对照 .结果显示 ,CaMBP 10在植物中的分布特点与文献报道的CaM的分布特点相一致 ,提示CaMBP 10可能是在蛋白水平上对CaM进行区域化和可用性调节     

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

ＳｉｎｃｅｔｈｅｆｉｒｓｔｒｅｐｏｒｔｂｙＢｅｎｎｅｔｔ[1]ｔｈａｔｍｕｌｔｉｐｌｅｃｈｌｏｒｏｐｌａｓｔｐｒｏｔｅｉｎｓｃｏｕｌｄｂｅｐｈｏｓｐｈｏｒｙｌａｔｅｄｂｙａｎｅｎｄｏｇｅｎｏｕｓｋｉｎａｓｅｗｈｉｃｈｗａｓｓｔｉｍｕｌａｔｅｄｂｙｌｉｇｈｔａｎｄｒｅｄｕｃｉｎｇａｇｅｎｔｓ,ｍｏｓｔｉｎｖｅｓｔｉｇａｔｉｏｎｓｒｅｇａｒｄｉｎｇｆｕｎｃｔｉｏｎａｎｄｒｅｇｕｌａｔｉｏｎｏｆｔｈｙｌａｋｏｉｄｐｒｏｔｅｉｎｐｈｏｓｐｈｏｒｙｌａｔｉｏｎｈａｖｅｂｅｅｎｃｏｎｃｅｎｔｒａｔｅｄｏｎｔｈｅ…     

质膜H~+-ATPase磷酸化对其活性的调节

 质膜Ｈ＋┐ＡＴＰａｓｅ磷酸化对其活性的调节＊凌启阆向左云刘华尚克进（南开大学生物化学及分子生物学系,天津３０００７１）ＲｅｇｕｌａｔｉｏｎｏｆＰＭＨ＋┐ＡＴＰａｓｅＡｃｔｉｖｉｔｙｂｙＩｔｓＰｈｏｓｐｈｏｒｙｌａｔｉｏｎＬｉｎｇＱｉ－ＬａｎｇＸｉａｎ...     

ABA、IAA和CaM对发育菜豆子叶质膜H~＋－ATPase活力的效应

以亲水性两相分配法从发育菜豆子叶制备的质膜制剂经冻融循环操作,部分膜微囊可转变成密闭的翻转型。取冻融４次的质膜微囊用于Ｈ＋－ＡＴＰａｓｅ试验表明,ＡＴＰａｓｅ活力为ＡＢＡ和ＣａＭ显著地激活,但受ＩＡＡ显著抑制;质子泵活力被ＡＢＡ显著促进,但为ＣａＭ显著抑制,ＩＡＡ对质子泵活力无显著效应。可以认为：ＡＢＡ促进发育菜豆子叶吸收光合同化物可能是通过促进质膜Ｈ＋－ＡＴＰａｓｅ活力,从而促进质子／蔗糖同向运输而获得;ＩＡＡ则可能对菜豆子叶的质膜Ｈ＋－ＡＴＰａｓｅ无显著效应。在激素信号传导途径中,ＣａＭ对质膜Ｈ＋－ＡＴＰａｓｅ活力可能无直接影响。     

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.     

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.     

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.     

生长素信号转导研究进展

长素的信号转导是一个复杂的网络系统,在信号的感知上,除了存在ABPI介导的膜上感知途径外,还有其他的感知途径。G蛋白参与诱导生长素信号的胞内传递,生长素信号转导的第二信使包括离子型第二信使、磷酯酶A2、脂活化蛋白激酶、MAPK和PINOIND等。AUX／IAA蛋白的泛素化降解在生长素反应中发挥关键性作用,ARF和AUX／IAA蛋白相互作用调节生长素响应基因的转录。