温度和底物对大豆液泡膜H~+-ATPase二级结构的影响

利用圆二色性光谱,检测了纯化的大豆液泡膜Ｈ＾＋－ＡＴＰａｓｅ在不同条件下蛋白二级结构的变化。液泡膜Ｈ＾＋－ＡＴＰａｓｅ的圆二色光谱对温度敏感,２５℃、３７℃分别保温１０分钟２０分钟,２０８ｎｍ和２２２ｎｍ双负峰变小,酶蛋白α－螺旋含量减少,与２５℃相比较,３７℃保温时酶蛋白构象的变化更为剧烈、迅速。不同磷酸数目的腺苷酸处理,液泡膜Ｈ＾＋－ＡＴＰａｓｅ的α－螺旋含量均降低,降低程度为ＡＤＰ＞ＡＭＰ＞     

大豆液泡膜H~+-ATPase泵质子活性的研究

研究了大豆液泡膜Ｈ＋－ＡＴＰａｓｅ泵质子特性。液泡膜Ｈ＋－ＡＴＰａｓｅ泵质子活性受ＮＥＭ、ＮＢＤ－Ｃｌ、ＤＣＣＤ和ＮＯ３－的抑制。泵质子活性由二价阳离子启动,其有效性依次为Ｆｅ２＋＞Ｍｇ２＋＞Ｍｎ２＋,它以ＡＴＰ为最适底物,ＡＤＰ为竞争性抑制剂;最适ｐＨ为７．０,最适温度为５０°Ｃ。     

大豆液泡膜H+-ATPase功能与构象关系的初步研究

大豆液泡膜V型H⁺-ATPase是ATPases中的一种,它在植物细胞的生长发育中有重要的作用.利用竹红菌乙素(HB）和KI这两种分别猝灭蛋白质疏水区域内源荧光和亲水区域内源荧光的荧光猝灭剂,在不同pH值、温度条件下对纯化的大豆液泡膜V型ATPase进行荧光猝灭实验,初步探讨了V型H⁺-ATPase的水解活性同其蛋白质折叠状态间的关系.研究表明,通过比较不同pH值、温度条件下蛋白质疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）,发现当环境pH值、温度偏离酶的最适pH值和温度时,蛋白质的内源荧光强度降低且疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）降低,说明伴随着酶的水解活性降低,蛋白质的折叠状态发生了变化.我们认为蛋白质在膜内的折叠状态变化是酶失活机制的一个重要方面,为植物的抗冻和抗盐研究提供了一定的参考.     

盐胁迫下无花果细胞质和液泡膜H^＋—ATPase活性对脯氨酸积累 …

盐胁迫降低无花果振荡培养细胞培养液ＰＨ添加质膜Ｈ＾＋－ＡＴＰａｓｅ活性抑制剂Ｎａ３ＶＯ４则抑制盐诱导的培养液ＰＨ下降,表明盐诱导培养液Ｈ下降主要是细胞质膜Ｈ＾＋－ＡＴＰａｓｅ活性增加的结果。ＮａＣｌ处理提高活体细胞质膜Ｈ＾＋－ＡＴＰａｓｅ活性,而降低膜微囊Ｈ＾＋－ＡＴＰａｓｅ活性,培养液中添加Ｎａ３ＶＯ４ ５０μｍｏｌ／Ｌ完全抑制盐胁迫下无花果细胞游离脯氨酸只累,但添加更高浓度Ｎａ３ＶＯ４,则提高     

盐胁迫下钙对大麦根系质膜和液泡膜功能的保护效应（简报）

盐胁迫下外加钙可增强大麦根系质膜液泡膜Ｈ＾＋－ＡＴＰａｓｅ、液泡膜质子泵和Ｎａ＾＋／Ｈ＾＋逆向运输活性,促进根系对Ｋ＾＋的选择性吸收和运输,降低叶片的Ｎａ＾＋／Ｋ＾＋比。     

花生幼苗下胚轴质膜Ca2+-ATP酶及其对低温胁迫的反应

经６％－１２％ＤｅｘｔｒａｎＴ７０密度梯度离心,获得了纯度较高的７ｄ龄花生幼苗下胚轴质膜制剂,质膜Ｃａ＾２＋－ＡＴＰａｓｅ在反应系统不存在Ｍｇ＾２＋时,可正常表现水解ＡＴＰ的活性,但此活性明显低于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,Ｃａ＾２＋－ＡＴＰａｓｅ不受Ｎａ３ＶＯ４抑制,不被Ｋ＾＋激活,而被Ｃｌ＾－抑制,Ｃａ＾２＋－ＡＴＰａｓｅ的最适,ｐＨ不同于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,低温胁迫显著提高质膜Ｃ     

跨膜Ca~(2+)梯差对大豆下胚轴质膜H~+-ATPase活力的影响

采用两相法得到高纯度封闭的大豆下胚轴质膜微囊,研究了跨膜Ｃａ２＋梯差对质膜Ｈ＋－ＡＴＰａｓｅ质子转运和ＡＴＰ水解活力的影响。结果表明,在１０００：０．１,１０００：０．５,１０００：１及１０００：１０（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）几种梯差下,随着跨膜钙梯差的减小,质膜Ｈ＋－ＡＴＰａｓｅ质子转运活力逐步降低。然而,上述几种梯差对Ｈ＋－ＡＴＰａｓｅ水解活力的影响却很小。进一步研究发现,１０００：０．１及１０００：１（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）两种梯差对Ｋｍ值没有影响,但Ｋ＋对Ｈ＋－ＡＴＰａｓｅ的激活作用在两种梯差下存在显著差别。ＭＣ５４０荧光、ＤＰＨ荧光偏振结果表明,跨膜钙梯差影响着膜脂的聚集状态和流动性。本文对跨膜Ｃａ２＋梯差对于大豆下胚轴质膜Ｈ＋－ＡＴＰａｓｅ水解与质子转运活力影响的可能机制进行了讨论。     

水稻幼苗寒害产生的生化机制研究

诱导性冷失活是Ｖ型ＡＴＰａｓｅ的一种共同特性。用荧光光谱技术以水稻液泡膜Ｈ＾＋－ＡＴＰａｓｅ为研究对象,深入探讨了水稻幼苗冷失活现象的机制。研究表明,能引起较大程度冷失活现象的物质,在往往先对膜脂有序性产生影响,通过对多种阳离子、阴离子、离子螯合剂导致冷失活能力和导致膜脂有序性变化能力间的比较,都能说明这一点,可以认为膜脂状态变化是冷失少机制发生的一个重要方面,也是植物早期寒害产生的原因之一。     

消炎痛对猪胃H~＋／K~＋－ATP_（ase）抑制的动力学

消炎痛作为一种可引起胃粘膜急性病变的药物,用分离提纯的猪胃Ｈ＋／Ｋ＋－ＡＴＰａｓｅ证明,它可以显著的抑制此酶的活力,０．１ｍｇ／ｍＬ时即可抑制酶活力２７％,０．５ｍｇ／ｍＬ时可抑制全部活力,其Ｋ（０．５）为０．１８ｍｇ／ｍＬ。消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制随３０℃时预保温时间的延长而加剧,１０ｍｉｎ预保温可抑制总活力的５０％。消炎痛并不影响Ｈ＋／Ｋ＋－ＴＡＰａｓｅ的转换温度（３９℃）以及最适ｐＨ（约ｐＨ７．５）,但酸性条件下消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ抑制比碱性条件下强烈。在我们的实验条件下,消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制与Ｈ＋／Ｋ＋－ＡＴＰａｓｅ量成正比,它不影响酶的Ｋｍ值（０．１１ｍｍｏｌ／Ｌ）,而是显著降低Ｖｍａｘ,因而它是此酶的可逆性非竞争性抑制剂,其Ｋｉ为０．３２ｍｍｏｌ／Ｌ。     

水分胁迫下钙螯合剂与CPZ抑制剂对棉花根和下胚轴两种ATPase…

水分胁迫下棉花根和下胚轴质膜Ｈ＾＋－ＡＴＰａｓｅ和Ｃａ＾２＋－ＡＴＰａｓｅ活力,表现Ｋｍ值以及Ｖｍａｘ降低。－０．３ＭＰａ和－１．１ＭＰａ胁迫下质膜ＡＴ－Ｐａｓｅ活力随时间延长分别呈“Ｖ”字形变化和下降趋势。钙螯合剂、ＣａＭ抑制剂对棉花根和下胚轴质膜ＡＴＰａｓｅ活性有明显的抑制效应,抑制程度为－１．１ＭＰａ大于－０．３ＭＰａ大于对照。     

A sensitive fluorescence quenching method for the detection of tartrazine with acriflavine in soft drinks

In this work, a simple, rapid, sensitive, selective spectrofluorimetric method was applied to detect tartrazine. The fluorescence of acriflavine could be efficiently quenched by tartrazine. The method manifested real time response as well as presented satisfied linear relationship to tartrazine. The linear response range of tartrazine (R² = 0.9995) was from 0.056 to 5 μmol L^?1. The detection limit (3σ/k) was 0.017 μmol L^?1, indicating that this method could be applied to detect traces of tartrazine. The accuracy and precision of the method was further assured by recovery studies via a standard addition method, with percentage recoveries in the range of 96.0% to 103.0%. Moreover, a quenching mechanism was investigated systematically by the linear plots at varying temperatures based on the Stern–Volmer equation, fluorescence lifetime and UV–visible absorption spectra, all of which proved to be static quenching. This sensitive, selective assay possessed a great application prospect for the food industry owing to its simplicity and rapidity for the detection of tartrazine.     

超氧阴离子诱导的叶绿素荧光猝灭

分别通过黄嘌呤(X)与黄嘌呤氧化酶(XO)反应和甲基紫金(MV)的作用,观察了O·-2诱导莴苣叶绿体的叶绿素荧光猝灭过程.结果表明,O-·2的产生明显使光化学猝灭(qP)和非光化学猝灭(qN)增加.叶绿体内SOD被DDC抑制后,X+XO诱导的叶绿素荧光猝灭过程中,qP下降,qN上升;MV诱导的叶绿素荧光猝灭过程中,qP上升幅度不大,qN增加不明显.当碳代谢被碘乙酰胺(JAA)抑制后, qP下降,qN上升.解偶联剂NH4Cl增加质子跨类囊体膜的通透性,导致qP增加和qN降低,加入MV后qP和qN增加不明显.分析认为,-·2的产生和及时被清除对保持光合电子传递和增加跨膜ΔpH有很重要的作用,有利于叶绿体吸收的光能得到转化和耗散,在一定程度上减轻过量光能引起的光抑制损伤.     

Chlorophyll fluorescence performance of sweet almond [Prunus dulcis (Miller) D. Webb] in response to salinity stress induced by NaCl

One-year old sweet almond (Prunus dulcis) seedlings were submitted to four levels of salt stress induced by NaCl, namely 0.3, 0.5, 0.7, and 1.0 S m⁻¹. Effects of salt stress on a range of chlorophyll (Chl) fluorescence parameters (Chl FPs) and Chl contents were investigated in order to establish an eco-physiological characterization of P. dulcis to salinity. Salt stress promoted an increase in F₀, F_s, and F₀/F_m and a decrease in F_m, F′_m, F_v/F_m, q_P, ΔF/F′_m, F_v/F₀, and UQF_(rel), in almost all Chl fluorescence yields (FY) and FPs due to its adverse effect on activity of photosystem 2. No significant changes were observed for quenchings q_N, NPQ, and q_N(rel). The contents of Chl a and b and their ratio were also significantly reduced at increased salt stress. In general, adverse salinity effects became significant when the electric conductivity of the nutrient solution (EC_n) exceeded 0.3 S m⁻¹. The most sensitive salt stress indicators were F_v/F₀ and Chl a content, and they are thus best used for early salt detection in P. dulcis. Monitoring of a simple Chl FY, such as F₀, also gave a good indication of induced salt stress due to the significant correlations observed between the different Chl FYs and FPs. Even essential Chl FYs, like F₀, F_m, F′_m, and F_s, and mutually independent Chl FPs, like F_v/F₀ and q_P, were strongly correlated with each other.     

Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components – calculation of qP and Fv-/Fm-; without measuring Fo-;

Imaging of chlorophyll a fluorescence from leaves has enabled the spatial resolution of the fluorescence parameter, F/Fm-;. Although this parameter provides a reliable estimate of photosynthetic efficiency under most conditions, the extent to which this efficiency is defined by (i) competition with other energy-dissipating processes operating at photosystem II and (ii) by processes on the reducing side of photosystem II, such as carbon assimilation, requires the use of additional parameters. Of particular value are qP, which quantifies the photochemical capacity of photosystem II, and Fv-;/Fm-;, which quantifies the extent to which photochemistry at photosystem II is limited by competition with thermal decay processes. Imaging of both qP and Fv-;/Fm-; requires measurement of Fo-; (the minimum fluorescence yield in the light-adapted state), which cannot be imaged with existing systems. In this paper, a method is described which estimates Fo-; through a simple equation involving the minimum fluorescence yield in the dark-adapted state (Fo), the maximum fluorescence yield in the dark-adapted state (Fm), and the maximum fluorescence yield in the light-adapted state (Fm-;). This method is tested here, through comparison of measured and calculated values of Fo-;. An example of the application of this method to analysis of photosynthetic performance in leaves, from images of chlorophyll a fluorescence, is also presented.     

Probing the structural diversities of long alpha-neurotoxins by fluorescence quenching studies

Trp fluorescence of Ophiophagus hannah neurotoxins (Oh-4, Oh-6A, Oh-7, and Oh-8) and Bungarus multicinctus -bungarotoxin was quenched by acrylamide and iodide. Acrylamide quenching studies indicated that the degree of exposure of Trp residues in the neurotoxins followed the order Oh-8 > Oh-7 > Oh-6A > Oh-4 > -bungarotoxin, as did the accessibility for iodide. These results reveal that the exposed degree of Trp residues and the microenvironment surrounding Trp residues in the neurotoxins differ, even though their Trp residues and positively charged residues are located at the same or homologous positions. In contrast to unfolded Oh-4, Oh-6A, Oh-7, and -bungarotoxin, unfolding of Oh-8 by reduction and S-carboxymethylation caused a notable decrease in the susceptibility of their Trp residues for iodide. These observations support the view that the side chains of Trp residues and positively charged residues in their native structure do not point toward the same spatial positions. Computer models of the neurotoxins are in good agreement with this proposition. These results elucidate why the conserved Trp residues and cationic groups do not always play the same roles in the biological activities of the neurotoxins.     

Non-photochemical quenching of Fo in leaves is emission wavelength dependent: consequences for quenching analysis and its interpretation

The effects of light-induced non-photochemical quenching on the minimal F_o, and variable F_v, fluorescence emissions at 690 and 730 nm in leaves were determined. Non-photochemical quenching of F_o, but not F_v, was found to be dependent upon the wavelength of emission, and was greater at 690 nm than at 730 nm. For emission at 730, compared to at 690 nm, approx. 30% of F_o was not affected by non-photochemical quenching processes in leaves of C3 plants; in maize leaves this was found to be approx. 50%. The data indicate that a substantial proportion of the pigments contributing to F_o emission at 730 nm are not quenched by light-induced, non-photochemical quenching processes and that there are large differences in the pigment matrices contributing to F_o and F_v emissions at 730 nm, compared to those at 690 nm. These findings have important implications for the accurate estimation and interpretation of non-photochemical quenching of fluorescence parameters and their use in the calculation of photochemical efficiencies in leaves. Measurements of fluorescence emissions at wavelengths above 700 nm are likely to give rise to significant errors when used for determinations of photochemical and non-photochemical quenching parameters.     

两种杂交杨叶绿素荧光特性及光能利用

比较研究了伊犁地区两种杂交杨伊犁杨1号(Populus deltaids‘cv-64’ (P64))和伊犁杨小叶杨(P. simonii canaden×P. russkii-9(Jia))对太阳辐射光能的利用和耗散特性。两种杂交杨光合速率(P_n)的日变化均呈现双峰型, 高光量子通量密度(PFD)阶段P_n达到20.1%的差距; 实际最大光化学猝灭ΦPSII日变化均呈“U”型, 于16:30时, P64的ΦPSII达到最低值, 而Jia的值于14:30时达到最低(Jia>P64); 光合系统的闭合程度(L)在14:30时均出现一个短暂回落, 全天平均闭合程度无显著差异(p>0.05)。非光化学猝灭系数(NPQ)在16:30同时达到最大值(Jia>P64), 两者NPQ全天相差31.7%。叶片转化吸收太阳能热能耗散(E.D)和光化学反应转化的光能量(E.P)进行估算表明: 在PFD较低的环境条件下, 两种杂交杨将吸收的光能50%以上用于光化学猝灭; 在PFD较高的环境条件下, P64的E.P值比例大于Jia, 两者全天的E.P值没有太大的差异, P64的E.D值显著大于Jia的E.D值(p>0.01), 而P64的E.D值占全天转化能量的比例小于Jia。P64和Jia的E.P达到最大的估算值时, 其E.D也达到了最大。分析结果表明: 两种杂交杨对高光能形成不同的适应机制, P64利用更多的太阳能进行光化学猝灭反应, 而Jia利用更多的太阳能进行非光化学猝灭反应, 减缓强太阳辐射伤害; 两种杂交杨用于光化学能量分配的比例P64大于Jia, 而P_n值在强辐射阶段和全天平均值、累积值均出现Jia>P64。结果证明, 仅通过杂交杨本身叶绿体对光的荧光特性反应计算接收到的光能中有多少能量被利用与实际植物光合速率的转化的干物质存在一定的差异, 两种杂交杨光化学实际固定碳和转化光能的多少的内在关系需进一步的研究。     

Quenching of the fluorescence emitted by P695-682 at room temperature in etiolated illuminated leaves

Chlorophyll(ide) fluorescence emission decreases at room temperature during completion of protochlorophyll(ide) reduction. The process responsible for this quenching is parallel to the P_688-676 P_695-682 transition. It proceeds equally well in darkness and in the light. It consists in a decrease of the fluorescence yield of chlorophyll(ide) in P_695-682. Apparently, room temperature P_695-682 fluorescence is regulated by a conjunction of factors such as energy transfers and photobiochemical activities.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - CPI chlorophyll-protein-complex I - CPII chlorophyll-protein-complex II Aspirant du Fond National de la Recherche Scientifique, Belgium     

热锻炼对甘蓝幼苗叶片激发能分配的影响

以喜温凉的蔬菜甘蓝为试材,研究了热锻炼与对照甘蓝幼苗叶片光合速率和叶绿素荧光参数对高温胁迫的响应．结果表明,叶片温度在25-35℃之间,热锻炼苗和对照苗叶片叶绿素可变荧光(Ｆv)、光化学猝灭(qP)、非光化学猝灭(qN)、PSⅡ化学效率(ФPSⅡ)没有明显的变化;当叶温高于35℃时。热锻炼苗的Fv、qP和中ФPSⅡ均明显高于对照,37℃时Fv、qP和ФPSⅡ分别比对照高53％、24％和86％;qN较对照低22％,尤其是与光抑制(光破坏)有关的qNs明显降低,以维持较高的高能态猝灭(qNf)耗散过剩激发能。保护PSⅡ反应中心不受破坏。减轻了光抑制,这与热锻炼幼苗叶片在高温下具有较高的光合能力是一致的。     

From Ca2+ in muscle contraction to IP3 receptor/Ca2+ signaling In memory of Setsuro Ebashi

The red fluorescent protein, DsRed, and a few of its mutants have been shown to bind copper ions resulting in quenching of its fluorescence. The response to Cu²⁺ is rapid, selective, and reversible upon addition of a copper chelator. DsRed has been employed as an in vitro probe for Cu²⁺ determination by us and other groups. It is also envisioned that DsRed can serve as an intracellular genetically encoded indicator of Cu²⁺ concentration, and can be targeted to desired subcellular locations for Cu²⁺ determination. However, no information has been reported yet regarding the mechanism of the fluorescence quenching of DsRed in the presence of Cu²⁺. In this work, we have performed spectroscopic investigations to determine the mechanism of quenching of DsRed fluorescence in the presence of Cu²⁺. We have studied the effect of Cu²⁺ addition on two representative mutants of DsRed, specifically, DsRed-Monomer and DsRed-Express. Both proteins bind Cu²⁺ with micromolar affinities. Stern-Volmer plots generated at different temperatures indicate a static quenching process in the case of both proteins in the presence of Cu²⁺. This mechanism was further studied using absorption spectroscopy. Stern-Volmer constants and quenching rate constants support the observation of static quenching in DsRed in the presence of Cu²⁺. Circular dichroism (CD)-spectroscopic studies revealed no effect of Cu²⁺-binding on the secondary structure or conformation of the protein. The effect of pH changes on the quenching of DsRed fluorescence in the presence of copper resulted in pK_a values indicative of histidine and cysteine residue involvement in Cu²⁺-binding.