弱直流电场对枯草芽孢杆菌生长和代谢活性的作用

枯草芽孢杆菌63501(Bacillus subtilis 63501)培养液在连续培养时分别通入不同强度的直流电场,研究了它们在持续直流电刺激下的生长曲线、pH曲线、ATP酶活性、胞内总蛋白含量、细胞膜通透性以及细菌微观形态变化。 结果表明,适宜的直流电能促进枯草芽孢杆菌增殖;枯草芽孢杆菌的ATP酶活性在0.045 5×10-3 A/cm2电流密度作用下,在对数生长前期、后期和稳定期分别比对照组同期提升1.9、3.5和3.8倍,表明适宜强度的电流增强了细菌细胞的代谢活性。 细菌通透性也有不同程度的提高,与透射电子显微镜观察到的细菌细胞通电后的形态变化具有一致性。     

大骨节病患儿红细胞膜骨架的变化

本文对大骨节病患儿红细胞膜骨架稳定性及红细胞ATP含量进行测定,结果:1.膜骨架蛋白主要组分血影收缩蛋白(Spectrin)与膜的结合程度下降,2.Spectrin本身的聚合状态有变化,3.~(31)P-NMR测试结果表明,大骨节病患者红细胞膜化学位移各向异性增加,提示膜骨架蛋白与膜脂静电作用增加,磷脂‘头部’运动自由度减少,4.大骨节病患儿红细胞膜内含能源物质ATP明显减少,5.大骨节病患儿红细胞硒含量明显低于对照。本文还对硒与红细胞膜骨架,低硒与大骨节病发病机理进行了讨论。     

分子动力学研究F1-ATP合酶对三磷酸腺苷的稳定和定位作用

F₁-ATP合酶通过与ATP之间建立广泛的相互作用,实现对ATP的位置进行精确的定位.这些相互作用为ATP的合成/水解创造了稳定的环境.理解这些相互作用是理解ATP的合成/水解机理的基础.我们通过分子动力学模拟方法研究这些相互作用,找出在稳定化过程中起到重要作用的残基.通过检测ATP和F₁-ATP合酶之间的非键相互作用,发现残基段158-164所形成的loop区域及残基R189, Y345对ATP存在显著相互作用.其中,该loop区域对ATP的三磷酸部分形成一个半包围结构,封闭活性位点区域,并通过氢键网络约束ATP三磷酸的运动,为ATP合成/水解创造稳定的环境.此外,关键残基Y345通过π-π叠加相互作用对ATP的碱基进行约束,但是ATP的碱基可以在平行于Y345芳香环的平面内进行滑动,我们推断这种滑动运动有利于促进ATP的水解.     

双偏振极化干涉技术实时研究三磷酸腺苷与其适配体相互作用

利用双偏振极化干涉(Dual polarization interferometry,DPI)测量技术实时研究了三磷酸腺苷(ATP)与其适配体(ATP-binding aptamer, ABA)间的相互作用.将单链ABA固定在DPI氮化硅芯片上,采用DPI技术实时监测ATP与固定的ABA的相互作用过程中敏感层的质量、厚度、密度的变化.通过详细分析敏感层质量变化,得到ATP与ABA间的结合速率常数 (ka=4.66 × 10.3 L/(mol·s)、解离速率常数(kd =1.70 × 10.-2·s.-1)、结合常数 (KA=2.7 × 10.5 L/mol)和解离常数(KD =3.7 × 10.-6 mol/L).通过测定敏感层质量、厚度和密度随ATP浓度的变化,分别建立了测定ATP的方法,检出限(LOD, 3σ)分别为0.22 μmol/L(质量变化)、0.14 μmol/L(厚度变化)、0.32 μmol/L(密度变化).本研究利用DPI技术揭示了ABA与ATP相互作用中结构变化的实时信息,构建了新型ATP传感器,用于实际血清样品中ATP的检测,结果令人满意.     

基于微磁珠分离技术的适配体实时定量PCR检测方法

利用适配体的识别能力和可扩增性, 构建了基于微磁珠分离技术的适配体实时定量聚合酶链式反应(PCR)检测方法. 通过微磁珠偶联的互补链与适配体序列之间的碱基配对结合, 有效除去溶液中未与靶分子结合的适配体序列, 采用实时定量PCR技术测定上清液中结合态的适配体序列浓度, 从而间接实现对靶分子的定量检测. 分别选取代表生物大分子和有机小分子的凝血酶和ATP作为检测对象, 验证了该方法的普适性. 研究结果表明, 在获取特异性适配体序列后, 仅需简单优化其互补链序列, 即可对超低含量的凝血酶和ATP进行准确定量, 检出限分别为50 pmol/L和5 μmol/L. 该方法具有同时适用于高特异性和高灵敏度地检测生物大分子和有机小分子的优势.     

多种精氨酸衍生物与ＡＴＰ作用及其催化ＡＴＰ水解的影响研究

分别使用荧光光谱法、 ~1H和~(31)P核磁方法对腺苷-5′-三磷酸(ATP)和多种L-精氨酸衍生物(L)的相互作用做了系统的研究, 得到了一些有意义的结果.在荧光滴定实验中, L对ATP有很强的荧光猝灭作用, 说明两者有较强的相互作用. ~1H和~(31)P核磁结果进一步证实了L与ATP的识别位点在ATP的嘌呤环和磷酸链上, 结合力为氢键和静电作用. 另外, 用~(31)P 核磁滴定的方法考察了L对ATP水解的影响, 发现不同的精氨酸衍生物对ATP水解的催化影响有很大的差异, 说明L对ATP的识别和催化其水解作用有较强的选择性. 此识别及催化作用的研究对深入理解精氨酸残基在ATP合酶中起到"触点"作用有一定的参考价值.     

Ag-ZnO/凹凸棒石复合光催化剂的制备及性能研究

以凹凸棒石(ATP)为载体,二水合乙酸锌、硝酸银为原料,采用溶胶凝胶法结合超临界干燥技术制备了Ag-ZnO/ATP复合光催化剂。利用XRD、SEM、TEM、UV-Vis对复合光催化剂的结构进行表征,并通过降解亚甲基蓝溶液评估光催化活性。研究结果表明:该方法制得的Ag-ZnO/ATP复合光催化剂颗粒均匀,分散性良好,具有较高的光催化活性。Ag负载量为3wt%,煅烧温度为400℃时制备的复合光催化剂,在紫外灯下降解亚甲基蓝溶液3h后,降解率达98. 2%,COD去除率为96. 8%。制备的复合光催化剂稳定性良好,三次循环回收后,亚甲基蓝溶液的降解率达89. 7%。     

基于有机小分子的三磷酸腺苷荧光传感器研究进展

三磷酸腺苷(ATP)是各种活细胞内普遍存在的一种高能磷酸化合物,在能量的储存,细胞呼吸作用和酶催化反应等生物活动过程中扮演着重要作用.因此,对于ATP在生物体内的研究至关重要.荧光检测技术具有操作方便、选择性好和灵敏度高等优点,设计合成高效的ATP荧光传感器是近年来生物化学和分析化学领域的研究热点.根据ATP荧光传感器的结构特点和识别原理,将ATP荧光传感器分为金属Zn(Ⅱ)作为键合位点型识别,其它金属离子作为键合位点型识别和静电或氢键作用型识别.基于有机小分子荧光传感器,综述了近年来国内外ATP荧光传感器在分子设计与应用方面的研究进展,并展望了其发展趋势.     

对氨基脲敏感的胺氧化酶生物功能化色谱的方法学研究

采用毛细管电泳法建立了对氨基脲敏感的胺氧化酶(SSAO)生物功能化色谱模型.将SSAO酶固载到一种仿细胞膜的载体--脂质体上,pH值对固定化酶稳定性影响的实验表明,pH 5.0是理想的固载条件;SSAO酶活性检测表明游离态和固定态的酶活性分别为0.0404和0.0347 mmol g-1 min-1.将有活性的固定化SSAO酶作为假固定相添加到磷酸盐缓冲液中(10 mmol/L, pH 5).随着固定化酶的浓度从0.004 g/L上升到0.856 g/L,其特异性底物苯甲胺的有效淌度从4.06×10-4 cm2/(V · s)下降到0.81×10 -4 cm2/(V · s).据此,建立的生物功能化色谱模型为研究生物分子间的特异性作用提供了新的技术方法.     

基于生物发光的高灵敏度三磷酸腺苷检测研究

在微生物检测和食品安全控制中需要对低浓度的三磷酸腺苷(ATP)做出快速准确的测定,基于ATP光学反应原理,设计了一种微量光学反应池,结合萤火虫素-萤火虫素酶发光技术,构建了一种用于现场检测的高灵敏度ATP光学传感器系统.传感器响应波长为550 nm,ATP生物发光反应适宜pH为7.8,在优化实验条件下,发光强度和ATP浓度在对数坐标下呈线性相关,检出限可达1×10-16 mol/L ATP,检测样品仅需30 μL,测量时间60 s.这种检测方法具有响应范围宽,测试简便,快速可靠等特点,适用于ATP现场快速检测,具有很好的应用前景.     

Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography

A method for the determination of adenosine triphosphatase (ATPase) activity of myofibrils of big head carp by using ion chromatography was introduced. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and orthophosphate (Pi) were separated completely. Recoveries for ATP, ADP and Pi were 98+/-5%, 97+/-4% and 98+/-5%, respectively. Pi liberated from ATP during reaction was monitored by ion chromatography using the suggested method. This method was applicable to the determination of myofibrils ATPase activity for quick quality evaluation of surimi.     

Biostimulation of Na,K-ATPase by low-energy laser irradiation (685 nm, 35 mW): comparative effects in membrane, solubilized and DPPC:DPPE-liposome reconstituted enzyme

OBJECTIVE: The aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of the different forms of the Na,K-ATPase. METHODS: Membrane-bound and solubilized (alphabeta)(2) form of Na,K-ATPase was obtained from the dark red outer medulla of the kidney and proteoliposomes of DPPC:DPPE and Na,K-ATPase was prepared by the co-solubilization method. Irradiations were carried out at 685 nm using an InGaAIP diode laser. RESULTS: The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/cm(2). However, with irradiation doses ranging from 32 to 40 J/cm(2), a 28% increase on the ATPase activity was observed while when using up to 50 J/cm(2) no additional enhancement was observed. When biostimulation was done using the solubilized and purified enzyme or the DPPC:DPPE-liposome reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/cm(2). With irradiation above these values (24 J/cm(2)) no additional increase in the activity was observed. These studies revealed that the biostimulation of ATPase activity from different forms of the Na,K-ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.     

An on-line method for determining ATPase bioactivity and its application to human umbilical vein endothelial cell membrane

Based on the determination of inorganic phosphate (the product of ATP dephosphorylation by ATPase), a method for on-line determination of bioactivity of ATPase on cell membrane is presented, in which on-line sampling of cell membrane and separation of inorganic phosphate is employed in a flow injection system. The method has been successfully applied to the investigation of variation of ATPase from human umbilical vein endothelial cell membrane; the results show that ATPase activity decreases with elapsed time studied, which may supply important information for the pertinent studies on cell membrane enzymes bioactivity.     

Some changes in the properties of dynein ATPase in situ and after extraction following heat treatment of cilia.

Glycerol-extracted cilia from Tetrahymena pyriformis were demembranated by treatment with Triton X-100 and then heated for up to 30 min at temperatures between 34-38 degrees C. Heat treatment caused an uncoupling of the ATPase from motility as indicated by an increase in ATPase activity and a loss of pellet height response. After heat treatment, the ATPase activity of the dynein in situ differed from that in unheated cilia as shown by an increased sensitivity to a lower temperature of assay (0 degrees C) and by a loss of the activation normally observed upon reaction with N-ethylmaleimide or p-phenylenedimaleimide. Upon extraction of the heat-treated cilia by Tris-EDTA, there was a large loss in ATPase activity so that the heat-treated cilia yielded a crude dynein fraction with a lower specific activity compared with that obtained from unheated controls. The difference was not due to a change in the amount of protein recovered or in the amount of ATPase activity which remained unextracted. Resolution of the crude dynein by sucrose density sedimentation indicated that activity was lost from both the 14S and 30S peaks but more so from the latter than from the former. Thus dynein in situ in cilia in which the ATPase has been uncoupled from motility by gentle heat treatment differs in several important respects from dynein inside unheated cilia.     

Calcium and magnesium ATPases of the spectrin fraction of human erythrocytes.

Using a rapid method of preparation, spectrin has been isolated from human erythrocytes and its ATPase activity investigated. The ATPase activity with calcium has two distinct components, one with optimal activity when calcium and ATP are of equal concentration (low-Ca-ATPase) and another which is activated above 1 mM CaCl2 and is maximal at 100 mM CaCl2. There is also a Mg-ATPase with maximal activity at 10 mM MgCl2. The high-Ca-ATPase of spectrin, but not the low-Ca-ATPase, is inhibited by magnesium, while the Mg-ATPase is inhibited by Ca in excess of ATP. None of these activities exhibits the calcium-stimulated magnesium-dependent activity characteristic of the red cell calcium pump.     

Solvothermal Synthesis of Multiple Dihydropyrimidinones at a Time as Inhibitors of Eg5

Solvothermal synthesis of multiple dihydropyrimidinones at a time has been developed in inexpensive and green bio-based solvent lactic acid without any additional catalysts or additives. By this method, thirty new dihydropyrimidinone derivatives were synthesized in two batches and characterized. All of the compounds were screened by Eg5 motor protein ATPase assay, and the positive compounds were tested against the Caco-2 cell line, HeLa cell line, L929 cell line and T24 cell line in vitro. Among them, compound C9 exhibited the best inhibitory activity against motor protein ATPase with an IC₅₀ value of 30.25 μM and significant cytotoxic activity in the micromolar range against the cells above. The Lineweaver–Burk plot revealed that compound C9 was a mixed-type Eg5 inhibitor. A molecular modeling study using the Discovery Studio program was performed, where compound C9 exhibited good binding interaction with Eg5 motor protein ATPase, and this was consistent with the attained experimental results.     

Characteristics of the H(+)-translocating adenosine triphosphatase of Vibrio parahaemolyticus

We have characterized H(+)-translocating adenosine triphosphatase (ATPase) in membrane vesicles of Vibrio parahaemolyticus. The ATPase required high concentrations (about 0.5 M) of Na2SO4 (or other salts) for its maximum activity. Magnesium ion stimulated the ATPase activity, but Ca2+ did not. The activity of ATPase was inhibited by tetrachlorosalicylanilide, an H+ conductor, but not by another H+ conductor, carbonylcyanide-m-chlorophenylhydrazone. The activity was strongly inhibited by dicyclohexylcarbodiimide or Zn2+, and partially inhibited by azide, but not at all by vanadate.     

A Multiple Enzyme Method for Maasurement of K,Na ATPase

Abstract

A multiple enzyme method for spectrophotometrical measurement of K, Na ATPase activity is described. The method includes three enzymatic steps: Hydrolyses of ATP by ATPase, conversion of inosine to hypoxanthine and ribose -1- phosphate by purine nucleoside phosphorylase and finally xanthine oxidase mediated oxidation of hypoxanthine to xanthine with consequent formation of formazan from a tetrazolium salt. The phospholytic cleavage of inosine in the medium is dependent of an ATPase- phosphate complex.     

Effect of thiourea and substituted thioureas on dynein ATPase and on the turbidity response of Tetrahymena cilia.

The effects of thiourea and of several substituted thioureas -- phenylthiourea, alpha-naphtylthiourea, metiamide, and burimamide -- on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of beta-mercaptoethanol or dithiothreitol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (approximately 1 microM) concentrations of ATP and, less effectively, by AMP-PNP, but not by AMP-PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP-PNP. The thioureas inhibit the ATP-induced decrease in turbidity (measured as delta A350) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of beta-mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice-extracted axonemes is not changed by treatment with phenylthiourea or metiamide. These observations indicate that the thioureas react with at least two sets of SH or S--S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.     

Studies of the oligomycin-sensitive ATPase from yeast mitochondria. Reconstitution of ATP-32Pi exchange in the presence of phospholipids.

A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin- and uncoupler-senstitive ATP-32Pi exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase-phospholipid-cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 A were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex.