稀土离子对钙调蛋白与单克隆抗体分子识别的影响

分别采用酶联免疫吸附(ELISA)法和荧光标记技术比较了Ca²⁺,La³⁺,Eu³⁺和Yb³⁺离子对钙调蛋白与单克隆抗体2C3之间分子识别的影响.结果表明,金属离子与钙调蛋白作用后会诱导其发生不同的构象变化,并进一步影响到钙调蛋白与单克隆抗体2C3分子之间的结合强度.当钙调蛋白分别与La³⁺,Eu³⁺,Yb³⁺作用后,它与单抗2C3分子之间的解离常数为(26.8±2.5),(21.8±3.4)和(64.8±5.1)nmol/L,而结合Ca²⁺前后的钙调蛋白与单抗分子的解离常数分别为(177.2±2.8)和(157±4.2)nmol/L.这一结果表明,稀土离子诱导钙调蛋白发生的构象变化明显不同于钙离子的作用,这种差异可能是稀土与钙离子对钙调蛋白调控作用表现出差别的原因.     

一种钙/钙调蛋白依赖性的蛇毒磷脂酶A_2

分别研究了钙离子和三价稀土离子对白眉蝮蛇 (Agkistrodon blomhoffii Ussurensis)蛇毒磷脂酶 A2(PLA2 )活性的影响以及钙调蛋白对它的激活作用 .实验结果表明 ,PLA2 的活性对钙离子表现出依赖性 ,钙调蛋白能够激活该蛇毒 PLA2 ,钙调蛋白的拮抗剂三氟甲基吩噻嗪 (Trifluoperazine)能够完全抑制它对 PLA2的激活作用 .三价稀土离子 La3 、Eu3 、Dy3 、Yb3 对该 PLA2 的活性表现出抑制作用 ,其中离子半径较大的La3 和 Eu3 对酶活的抑制程度要小于半径较小的 Dy3 和 Yb3 .     

稀土离子对钙调蛋白与蜂毒素作用的影响

分别用钙调蛋白和蜂毒素的内源荧光光谱以及铽离子的敏化荧光光谱考察了铽离子对钙调蛋白构象变化以及对钙调蛋白与蜂毒素相互作用的影响 .结果表明 ,铽离子首先结合在钙调蛋白的第Ⅰ和第Ⅱ位点 ,铽离子不影响钙调蛋白与蜂毒素的相互作用 ,蜂毒素与钙调蛋白作用后不影响铽离子在钙调蛋白上的键合顺序 .傅里叶变换红外光谱结果表明三价的镧离子与钙调蛋白作用使钙调蛋白的α螺旋结构增加 ,β折叠结构减少 ,与钙离子对它的二级结构影响相类似 .稀土离子在钙调蛋白 -蜂毒素复合体系中主要与钙调蛋白作用 .     

稀土离子和钙调蛋白对乳酸脱氢酶活性的影响

分别研究了三价稀土离子、稀土离子和钙调蛋白(CaM)的复合物以及作为稀土离子配体的二乙三胺五乙酸(DTPA)及其衍生物二乙三胺五乙酸-双二甲酰胺(DTPA-BDMA)和二乙三胺五乙酸-双(异烟肼)(DTPA-BIN)对乳酸脱氢酶(LDH)活性的影响.结果表明稀土离子浓度小于3μmol@L-1时对酶活性具有轻微的激活作用,离子浓度增大到6μmo1@L-1后则开始表现出抑制作用;钙调蛋白可以缓解这种抑制作用;二乙三胺五乙酸及其衍生物在浓度超过5μmol@L-1时对酶活性均有抑制,这种抑制作用在加入外源的钙调蛋白后也得到缓解.     

一种钙/钙调蛋白依赖性的蛇毒磷脂酶A2

稀土离子;活性;一种钙/钙调蛋白依赖性的蛇毒磷脂酶A2     

稀土及其配合物对蛇毒磷脂酶A_2活性的影响

分别研究了三价稀土离子 (La3 + ,Eu3 + ,Dy3 + ,Yb3 + )、二乙三胺五乙酸及其衍生物二乙三胺五乙酸 -双二甲酰胺 ,二乙三胺五乙酸 -双 (异烟肼 )与稀土离子的配合物以及Tb -谷氨酰胺配合物对蛇毒磷脂酶A2 活性的影响 .浓度低于 <3μmol/L的稀土离子可以激活磷脂酶A2 ,浓度大于 5 μmol/L后稀土离子对酶活性表现出抑制作用 ;外源Ca2 + 离子的加入可以缓解稀土离子对酶活性的抑制作用 ,表明稀土离子和钙离子是竞争性地结合在酶的活性部位 ;稀土离子和二乙三胺五乙酸及其衍生物的配合物对酶活性没有明显影响 ;Tb -谷氨酰胺在浓度大于 10 μmol/L后开始抑制酶的活性     

抗羟甲芬太尼单克隆抗体和抗羟甲芬太尼独特型抗体的特性研究

本文采用羟甲芬太尼免疫的BALB/c小鼠脾脏细胞与小鼠骨髓瘤细胞NS-1融合,获得分泌抗羟甲芬太尼抗体的单克隆杂交瘤细胞株。该株细胞分泌的抗羟甲芬太尼单克隆抗体与羟甲芬太尼结合的亲合力为3.65±0.59×10~7 l/mol。羟甲芬太尼类似物3-甲基芬太尼、β-羟基芬太尼和芬太尼与抗羟甲芬太尼单克隆抗体有部分交叉反应,而DAGO,纳洛酮和双氢依托啡与抗羟甲芬太尼单克隆抗体无交叉反应。应用纯化的抗羟甲芬太尼单克隆抗体免疫家免,获得高滴度的兔抗羟甲芬太尼独特型抗体。抗羟甲芬太尼独特型抗体与抗羟甲芬太尼单克隆抗体结合反应有剂量关系,并呈可饱和性。抗羟甲芬太尼独特型抗体能竞争抑制抗羟甲芬太尼单克隆抗体与原始抗原羟甲芬太尼的结合反应。受体结合分析表明抗羟甲芬太尼独特型抗体能与放射性配体[~3H]羟甲芬太尼和[~3H]DAGO竞争结合大鼠脑匀浆膜蛋白上的阿片受体。在离体生物检定实验中抗羟甲芬太尼独特型抗体还具有类似羟甲芬太尼样作用,能呈剂量依赖地抑制电场刺激引起的小鼠输精管的收缩。以上结果提示抗羟甲芬太尼独特型抗体能与羟甲芬太尼竞争结合抗羟甲芬太尼单克隆抗体上的同一结合位点,具有羟甲芬太尼的内影像结构,并能与μ阿片受体结合,呈阿片受体激动剂样作用。     

三价镨钕与α-羟基羧酸配合物的理论模拟

在实验工作的基础上, 探索稀土配合物结构理论计算的可能性, 并利用分子模型（ＭＭ＋ ）、ＩＮＤＯ等理论方法计算了α羟基羟酸配体对三价镨钕离子的分离作用, 得到三价镨钕配合物分子的势能差与三价镨钕离子分离因数之间存在同步升降的变化趋势; 计算了配位键的特性以及配体的空间位阴效应; 指出了用分子模型来确定和设计对三价镨钕离子具有高分离因数配体结构的可能性。在实验工作的基础上, 探索稀土配合物结构理论计算的可能性, 并利用分子模型（ＭＭ＋ ）、ＩＮＤＯ等理论方法计算了α羟基羟酸配体对三价镨钕离子的分离作用, 得到三价镨钕配合物分子的势能差与三价镨钕离子分离因数之间存在同步升降的变化趋势; 计算了配位键的特性以及配体的空间位阴效应; 指出了用分子模型来确定和设计对三价镨钕离子具有高分离因数配体结构的可能性。     

钙黄绿素对Yb(Ⅲ)近红外发光的敏化作用研究

近红外发光可用于荧光免疫分析、激光系统及光信号放大等方面，三价镧系离子配合物的近红外发光的研究引起了人们的兴趣。近红外发光稀土离子的发射能级低，特别容易受到溶剂分子的碰撞而产生荧光猝灭作用，通过优化配体的结构，提高配体对中心稀土离子的配位作用，尽     

冠醚取代蒽醌超分子体系的设计与合成及分子内能量转移的研究

大环冠醚由于其自组装性能及分子识别能力而引起人们广泛的重视.近来,冠醚又成为在超分子体系中用于建构主体分子的一种重要的建造单元^[1~4].Costa^[5]等曾报道苯并冠醚与被识别的稀土离子铕(Ⅲ)与铽(Ⅲ)之间的能量传递过程,选择性激发冠醚主体观察到稀土离子的发光.由于二氮杂冠醚与稀土离子能形成更稳定的配合物^[6].我们利用了冠醚分子的分子识别能力及蒽醌分子的光敏性,设计合成了一种新的氮杂冠醚取代蒽醌分子(图1)(以下文中用代号AQ-CW表示),并以该分子作为主体分子,以稀土离子作为客体构成超分子体系,研究超分子体系内的能量转移过程.     

Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.     

Calcium ion fluorescence detection using liposomes containing Alexa-labeled calmodulin

This paper describes the preparation and characterization of calcium ion sensitive fluorescent liposomes and their application for the determination of calcium ions in aqueous samples. Calmodulin (CaM), a calcium ion-binding protein labeled with the fluorophore Alexa-488 is embedded in the membrane of unilamellar liposomes. Upon calcium ion binding, calmodulin undergoes a conformational change that exposes its hydrophobic core and affects the fluorescence intensity of the attached fluorophore. Characterization studies of Alexa-CaM-containing liposomes reveal that embedding calmodulin molecules in the bilayer membrane of liposomes extends the lifetime of the calcium ion binding activity of calmodulin by about fourfold compared to the lifetime of its calcium-binding activity in free solution. Moreover, the calcium ion response of Alexa-CaM-containing liposomes is about threefold higher than the calcium ion response of Alexa-CaM in solution. The improvement in the calcium ion detection properties is attributed to the interaction between calmodulin, a membranal protein, and the hydrophobic phospholipids of the liposomes. The analytical properties of the calcium ion sensitive fluorescent liposomes are discussed.     

Metal ions-induced conformational change of P23 by using TNS as fluorescence probe

In 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), pH 7.4, 25 degrees C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P23) induced by metal ions were investigated using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb3+ has more larger influence than Ca2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca2-loaded or Tb2-loaded P23 were obtained, K (Ca2-P23+TNS)=(7.49+/-0.88)x10(5) mol-1 L, K (Tb2-P23+TNS)=(8.24+/-0.49)x10(5) mol-1 L.     

协同发光效应及其分析应用研究

在研究共存元素对钐、铕、铽和镝配合物荧光发射的影响时,人们曾分别观察到这些配合物的荧光强度不但不随某些共存稀土和非稀土离子浓度的增加而降低;而是在一定浓度范围内随共存离子浓度的增加,配合物的荧光发射大大增强,利用这种效应可大大提高这类配合物的荧光分析灵敏度,我们采用自己组装的激光诱导荧光光谱测定装置,选取铕(钐)-     

The use of ESI-MS to probe the binding of divalent cations to calmodulin

Proteins have evolved with distinct sites for binding particular metal ions. This allows metalloproteins to perform a myriad of specialized tasks with conformations tailor-made by the combination of its primary sequence and the effect on this of the ligated metal ion. Here we investigate the selectivity of the calcium trigger protein calmodulin for divalent metal ions. This ubiquitous and highly abundant protein exists in equilibrium between its apo and its holo form wherein four calcium ions are bound. Amongst its many functions, calmodulin modulates the calcium concentration present in cells, but this functional property renders it a target for competition from other metal ions. We study the competition posed by four other divalent cations for the calcium binding sites in calmodulin using electrospray ionization mass spectrometry (ESI-MS). We have chosen two other group II cations Mg²⁺, Sr²⁺, and two heavy metals Cd²⁺, Pb²⁺. The ease with which each of these metals binds to apo and to holo CaM[4Ca] is described. We find that each metal ion has different properties with respect to calmodulin binding and competition with calcium. The order of affinity for apo CaM is Ca²⁺ ≫ Sr²⁺ ∼ Mg²⁺ > Pb²⁺ ∼ Cd²⁺. In the presence of calcium the affinity alters to Pb²⁺ > Ca²⁺ > Cd²⁺ > Sr²⁺ > Mg²⁺. Once complexes have been formed between the metal ions and protein (CaM:[xM]) we investigate whether the structural change which must accompanies calcium ligation to allow target binding takes place for a given CaM:[xM] system. We use a 20 residue target peptide, which forms the CaM binding site within the enzyme neuronal nitric-oxide synthase. Our earlier work (Shirran et al. 2005) [1] has demonstrated the particular selectivity of this system for CaM:4Ca²⁺. We find that along with Ca²⁺ only Pb²⁺ forms complexes of the form CaM:4M²⁺:nNOS. This work demonstrates the affinity for calcium above all other metals, but also warns about the ability of lead to replace calcium with apparent ease.     

Progress on Detection of Metals Ions by Functional Nucleic Acids Biosensor

Functional nucleic acids are natural or artificial nucleic acid sequences with specific functions and special structures. A part of metal ions are essential trace elements of human health, but excessive metal ions will be harmful to human health. The functional nucleic acids are widely used for detection of metal ions because of its advantages such as easy modification, low price, high stability and strong specificity. This paper detailed the interaction between functional nucleic acids and metal ions, mainly including cutting type, link type, metal ion-mediated base pairing, click chemistry type, conformational change type, and other types. The biosensors based on the combination of functional nucleic acid with different signal output were then introduced. Finally, the research significance and existing problems of functional nucleic acid for metal ion detection were discussed. The future development trends and applications of functional nucleic acid biosensor were prospected.     

氯化铕对大黄愈伤组织生长及超微结构的影响

以掌叶大黄的愈伤组织为材料，研究了不同浓度氯化铕对其生长的影响，并利用透射电镜技术研究了氯化铕对大黄细胞超微结构的影响。实验结果表明，低浓度氯化铕促进愈伤组织生长，高浓度则抑制生长，浓度达到１００ｍｇ／Ｌ则可导致大黄外植体全部死亡。氯化铕对大黄的促进作用可被钙调蛋白的拮抗剂氯丙嗪抑制。１０ｍｇ／Ｌ氯化铕可使大黄愈伤组织的细胞形态发生明显变异。     

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16- ion was the most abundant ion observed for the apo form and the 8- ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaMwith TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.