柱孢鱼腥藻的藻蓝蛋白亚基和F_(678)色素蛋白

柱孢鱼腥藻的藻蓝蛋白包含有两个亚基。β亚基具有578nm吸收峰和600nm荧光发射峰,分子量19.80±0.40KD,可表明β亚基仅有一个载色团。α亚基具有578nm和630nm吸收峰及645nm的荧光发射峰,分子量17.35±0.38,α亚基可能有两个载色团。别藻蓝蛋白有635nm和650nm吸收蜂及664nm的荧光发射峰。具藻胆体的类囊体膜从高盐浓度缓冲液移至低盐浓度缓冲液时表现678nm荧光发射峰,可能柱孢鱼腥藻存在的F_(678)色素蛋白相当于GLazer(1975)的别藻蓝蛋白B。     

鱼腥藻7120响应NaCl胁迫的光合特性

NaCl胁迫处理丝状蓝藻鱼腥藻7120后光合特性的变化表明;鱼腥藻7120的净光合放氧速率和呼吸速率随NaCl浓度的程式高而降低,且浓度低于0.4mol/LNaCl时的降幅比高于0.4mol/LNaCl时的降幅小,加入0.4%(W?V)的蔗糖后可提高盐胁迫后的鱼腥藻7120的光合放氧速率,吸收光谱测定结果表明盐胁迫没有改变鱼腥藻7120的光合色素组成,但导致藻胆蛋白的总含量降低,类胡萝卜素含量增加。低温荧光发射光谱测定表明盐胁迫后改变了光能在两个光系统之间的分配。由藻胆蛋白吸收的光能向光Ⅱ传递受阻。荧光动力学分析表明光系统Ⅱ的光化学效率随盐浓度的增加而降低。表现出与光合放氧速率的一致性。     

蚕豆和玉米叶绿体atpE基因表达产物的生物活性差异及次级结构分析

编码蚕豆和玉米叶绿体ATP合酶ε亚基的atpE基因分别在大肠杆菌中获得了高效表达 ,两种表达的ε亚基蛋白分别与来自蚕豆、玉米和菠菜的缺失ε亚基的CF1重组后 ,发现玉米的ε亚基蛋白在抑制CF1 ATP酶水解ATP、阻塞类囊体膜质子通道以及它促进光合磷酸化等方面均明显地强于蚕豆的ε亚基蛋白。该结果表明 :( 1)ε亚基对ATP合酶活性的调节作用与其同ATP合酶其他亚基间的亲和力大小密切相关 ;( 2 )ε亚基抑制CF1水解ATP和阻塞质子通道两个功能是呈正相关的。圆二色性 (circulardichroism)的分析结果表明 ,玉米CF1ε亚基的 4种二级结构比例为α 螺旋 2 2 .6% ,β 折叠 3 0 .6% ,β 转角 9.3 % ,无规则结构 3 7.7% ;蚕豆CF1ε亚基的 4种二级结构比例为α 螺旋 3 1.4 % ,β 折叠 2 2 .3 % ,β 转角 13 .8% ,无规则结构 3 2 .4 %     

多变鱼腥藻(Anabaena variabilis)藻胆体——类囊体膜光能传递的研究

多变鱼腥藻(Anabaena variabilis)藻胆体一类囊体膜的吸收峰位于678,624,490,438和418nm.当用580nm波长光激发藻胆体一类囊体膜中藻胆蛋白时,室温荧光峰位于662nm,在680nm附近有一肩;液氮温度荧光峰位于655,666,695和730nm.这说明藻胆蛋白捕获的光能能有效地传给叶绿素a.当用436nm波长光激发藻胆体一类囊性膜中叶绿素a时,室温荧光峰(?)于683nm;液氮温室荧光峰在730nm,另一小峰在695nm.表明叶绿素a捕获的光能不能传递给藻胆蛋白.藻胆体一类囊体膜放氧速率为245μmoleO_2/小时,毫克叶绿素,电境照片显示在类囊体膜上有大量藻胆体.用0.3M蔗糖,O.05M磷酸缓冲溶液洗藻胆体一类囊体膜,能使藻胆体与类囊体膜分开.对藻胆体与类囊体之间的光能传递进行了讨论.     

多变鱼腥藻(Anabaena variabilis)藻胆体——类囊体膜光能传递的研究

多变鱼腥藻(Anabaena variabilis)藻胆体一类囊体膜的吸收峰位于678,624,490,438和418nm.当用580nm波长光激发藻胆体一类囊体膜中藻胆蛋白时,室温荧光峰位于662nm,在680nm附近有一肩;液氮温度荧光峰位于655,666,695和730nm.这说明藻胆蛋白捕获的光能能有效地传给叶绿素a.当用436nm波长光激发藻胆体一类囊性膜中叶绿素a时,室温荧光峰(?)于683nm;液氮温室荧光峰在730nm,另一小峰在695nm.表明叶绿素a捕获的光能不能传递给藻胆蛋白.藻胆体一类囊体膜放氧速率为245μmoleO_2/小时,毫克叶绿素,电境照片显示在类囊体膜上有大量藻胆体.用0.3M蔗糖,O.05M磷酸缓冲溶液洗藻胆体一类囊体膜,能使藻胆体与类囊体膜分开.对藻胆体与类囊体之间的光能传递进行了讨论.     

柱孢鱼腥藻(Anabaena cylindkica)营养细胞和异形胞叶绿素蛋白复合体的比较研究

比较了柱孢鱼腥藻(Anabaena cylindrica)营养细胞和异形胞类囊体膜叶绿素蛋白复合体的种类和性质。以SDS增溶营养细胞类囊体膜和不连续聚丙烯酰胺电泳分离得到4个P700叶绿素a蛋白复合体,分别为GPIa、CPIb、CPIc和CPI;和1个系统Ⅱ叶绿素蛋白复合体CPa。相对迁移率小的4个复合体含有P700,呼收光谱红区吸收峰为675nm,液氮低温荧光发射光谱有728nm荧光发射峰。CPIa和CPI的分量子分别为205 和105千道尔顿。未见诸文献的CPIb和CPIc复合体的分子量介于CPIa和CPI之间。相对迁移率较大的CPa有着吸收光谱红区672nm吸收峰,液氮低温荧光发射光谱有687nm荧光发射峰,分子量为56千道尔顿。同时化学氧化还原差示光谱不表现P700吸收降低。柱孢鱼腥藻异形胞类囊体膜经SDS增溶和电泳分离得到2个系统Ⅰ叶绿素蛋白复合体,它们的吸收光谱特性和分子量大小相近于营养细胞分离的CPIa和CPI复合体。异形胞类囊体膜缺少系统Ⅱ叶绿索蛋白复合体。     

用毛地黄皂苷分离异形胞及其对光合和固氮的某些特性的影响

本文叙述一种从多变鱼腥藻(Anabaena variabilis)中分离异形胞的简易方法。这种新方法是用毛地黄皂苷和甘露醇的 TES 缓冲液处理藻丝,破碎营养细胞,并结合分级离心的方法获得异形胞。所分离异形胞的纯度,在显微镜下观察达到90%左右。当提供 ATP 和Na_2S_2O_4时,能够测到所分离异形胞的固氮活性,其最大速率是5.31毫微克分子 C_2H_2/10~6异形胞/小时,为整体藻丝活性的10%。这种比活性,在4小时内,甚至更长的时间内,保持不变。但是,在氢气下和照光条件下,分离的异形胞缺乏受光促进的固氮活性。分离的异形胞在77°K下波长为430nm 光激发的荧光光谱和完整藻丝的相比,它缺乏属于光合系统Ⅱ的685nm 和695nm 的荧光发射峰,而仅具有光合系统 I 的730nm 荧光发射峰。当提供 DCIP 和抗坏血酸时,被压碎的异形胞能够光还原甲基紫精,其活性为360消耗 O_2的微克分子/毫克叶绿素/小时。上述结果表明用毛地黄皂苷法分离的异形胞具有较完整的 DCIPH_2→MV 的 PSI 活性。     

高等植物叶绿体ATP合酶ε亚基的结构与功能

ε亚基是叶绿体ATP合酶最小的一个亚基,有阻塞ATP合酶的质子通道和抑制其水解ATP活力的两种功能.用定点突变和缺失等分子生物学方法对ε亚基的结构功能进行了研究,结果表明:ε亚基42位上的苏氨酸(Thr42)对维持其结构和功能都很重要.与大肠杆菌ATP合酶相比,叶绿体ATP合酶ε亚基C端和N端的氨基酸残基缺失对其结构功能的影响更为敏感.     

转基因鱼腥藻中hTNF-α基因表达与光合作用间的相关性研究

分析了培养光强对转基因鱼腥藻生长和hTNF-α基因表达的影响,以及转基因鱼腥藻IB02的光合放氧活性、光系统Ⅰ及光系统Ⅱ活性。发现光强对转基因鱼腥藻IB02的生长和hTNF-α基因表达都有促进;hTNF-α基因在鱼腥藻中的表达率与真正光合、光系统Ⅰ和光系统Ⅱ活性存在一定的联系。hTNF-α基因表达同时对宿主的光合放氧特性也产生了显著的影响,与正对照相比转基因藻光呼吸速率增强68%,饱和点降低66%,说明转基因鱼腥藻的代谢负荷增加,并在低光强下生长比野生型快。     

阳光紫外辐射对褐藻羊栖菜生长和光合作用的影响

为探讨经济褐藻羊栖菜对阳光紫外辐射变化的响应,我们在全波段阳光辐射(280－700 nm),去除UV-B辐射(320－700 nm)以及光合有效辐射PAR (400－700 nm)三种辐射条件下对其进行培养,测定了其光合作用与生长的变化。羊栖菜的生长是通过每两天测量一次藻体的湿重来测定的,光合放氧是用Clark型氧电极测定的,为了测定藻体叶绿素a和紫外吸收物质的含量,从250 nm到750 nm对羊栖菜的甲醇提取液进行扫描,叶绿素a的浓度用Porra的公式计算,紫外吸收物质的计算是根据Dunlap的方法先计算紫外吸收物质和叶绿素a的比率,然后乘以每单位藻体叶绿素a的含量。结果表明,当藻体接收较多的日辐射量时有较高的相对生长速率,当滤除UVR后,较高的太阳辐射也导致了较高的光合放氧。然而太阳紫外辐射能够抑制藻体的光合放氧和生长速率,降低叶绿素a的浓度,并且这种抑制作用随着辐射水平的升高而增强。此外,阳光紫外辐射也诱导产生了一定量的紫外吸收物质,但并不足以抵抗紫外辐射对藻体的伤害作用。     

Detection of chlorophyllide in chlorophyll-free plasma membrane preparations from Anacystis nidulans

Plasma and thylakoid membranes were isolated and purified from the cyanobacterium Anacystis nidulans. Spectrophotometric examination of acetone extracts gave major absorption bands resulting from carotenoids and chlorophyll a in plasma and thylakoid membranes, respectively. Only a very small absorption peak at 663 nm was detected in acetone extracts of plasma membranes which, in contrast to the corresponding peak from thylakoid membranes, could not be extracted into n-hexane; methanol, on the other hand, was effective with both plasma and thylakoid membranes. Aqueous membrane suspensions excited at 435 nm gave strong fluorescence emission at 662 nm for plasma membranes, but only a very small one for thylakoid membranes which had been adjusted to equal absorbance at 678 nm. Excitation spectra of the 668 nm fluorescence emission peak in acetone extracts of plasma and thylakoid membranes were strikingly different from each other. Finally, high performance liquid chromatography afforded clear-cut preparative separation of the two "chlorophyll-like" pigments in plasma and thylakoid membranes, respectively, and identification by comparison with retention characteristics known from the literature, together with a pure chlorophyll a standard. Our results indicate that the highly fluorescent and polar "chlorophyll-like" pigment in plasma membranes of Anacystis is a chlorophyll precursor, viz. chlorophyllide a.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

毕氏海蓬子光系统Ⅱ颗粒的分离与鉴定

采用去污剂TritonX-100增溶类囊体膜和高速离心的方法,首次分离和纯化了毕氏海蓬子的光系统Ⅱ（photosystemⅡ,PSⅡ）颗粒,通过光谱学和SDS-PAGE对其进行鉴定并与类囊体膜进行比较。室温吸收光谱结果表明,PSⅡ颗粒在蓝区的叶绿素（chlorophyll,ChOb和胡萝卜素类吸收峰为485nm,在红区的Ch1b吸收峰为655nm,这两个峰值均低于类囊体膜中的。77K荧光发射光谱结果表明,提取的PSⅡ颗粒基本不含光系统Ⅰ（photosystemⅠ,PSI）的低温荧光反射峰737nm。77K荧光激发光谱结果显示,海蓬子PSⅡ颗粒在470-485am之间的Ch1b 和胡萝卜素类的荧光发射峰明显低于类囊体膜的。这说明在PSⅡ中大部分的PSI已被除去。电泳结果显示,海蓬子PSⅡ颗粒缺少PSI反应中心蛋白质亚基PsaA和PsaB,这说明提取到的PSⅡ纯度较高,这为进一步研究毕氏海蓬子PSⅡ的结构与功能奠定基础。     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-beta-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

Biosynthesis of Chlorophyll Regulates Reconstruction of Thylakoid Membrane in Cyanobacterium Synechocystis sp. PCC 6803

By DNA recombination technology in vitro, ORF469- mutant of cynobacterium Synechocystis sp. PCC 6803 was constructed, in which the ORF469 fragment relative to the light-inde-pendent protochlorophyllide (Pchlide) reduction was deleted. In BG-11 medium with 5 mmol/L glucose, the mutant was grown in darkness with a brief period (10 min) of illumination everyday (light-activated heterotrophic growth, LAHG) for 2 weeks to delete chlorophyll (Chl). The 665 mn Chl peak was replaced by the 629 nm Pchlide peak in the absorption spectra of the methanol extracts. The absorption spectra of the intact cells showed only shoulder peak at 620 nm (representing phyco- biliprotein). The thylakoid membrane disappeared, but the amount of phycobilisome did not decrease. When the mutant was transferred from LAHG condition to continuous light illumination for 3 h, the absorbance at 665 nm became higher than that at 629 nm and two peaks at 620 nm and 440 nm,representing phycobiliprotein and Chi-protein complex respectively, appeared in the absorption spectra of the intact cells. Mter exposure to the light for 8 h, the thylakoid membrane was visible in the cells. And for 24 h, a shoulder peak was present at 680 nm in the absorption spectra of the intact cells. Meanwhile the absorption spectra of the methanol extracts had no difference from that of cells grown in the light. Mter 48 h, the shape of the absorption spectra of the intact cells became the same as that of cells grown in the light. The layers of thylakoid membranes were as clear as those of the cells grown in the light. The results indicated that the biosynthesis of chlorophyll regulates the reconstmction of thylakoid membrane rendering the Chl protein complex to play its functional role in photosystems.     

蓝细菌Synechocystis sp.PCC6803中叶绿素合成调控类囊体膜重建的研究

利用ＤＮＡ体外重组技术构建了蓝细菌Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变种ＯＲＦ４６９,它的染色体ＤＮＡ缺失ＯＲＦ４６９片段,该突变种在加入５ｍｍｏｌ／Ｌ葡萄糖的ＢＧ－１１培养基中经遮光培养２周后叶绿素全部消失,细胞甲醇提取液光谱中６６５ｎｍ处叶绿互峰消失,６２９ｎｍ处出现原叶绿素酸酯峰,完整细胞光谱仅存在６２０ｎｍ的藻胆蛋白肩峰,细胞的类囊体膜消失,但藻胆体颗粒并未减少;细胞培养物重     

Photosynthetic Adaptation to High Temperature Associated with Thylakoid Membranes of Synechococcus PCC7002

Photosynthetic adaptation to high temperature was investigatedin intact cells and isolated thylakoid membranes of the cyanobacterium,Synechococcus PCC7002. In intact cells, the thermal stabilityof photosynthesis and photosystem 2-mediated electron transportfrom H₂O to 1,4-benzoquinone changed in concert with growthtemperature. The photosystem 2-mediated electron transport fromH₂O to phenyl-1,4-benzoquinone showed greater thermal stabilityin thylakoid membranes isolated from cells which had adaptedto high temperature than in those from non-adapted cells. Enhancedthermal stability was also observed in the thylakoid membranesin the transport of electrons from H₂O to 2,6-dichlorophenolindophenolbut not in the transport of electrons from diphenylcarbazideto 2,6-dichlorophenolindophenol. These observations suggestthat oxygen-evolving sites acquire enhanced thermal stability,and that factors which are responsible for thermal stabilityremain in isolated thylakoid membranes. (Received October 30, 1992; Accepted December 18, 1992)     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

缺钼培养的蓝藻Anabaena 1720放氢的研究

培养液中缺钼时,蓝藻Anabaena 7120的放氢受到削弱,其削弱程度比固氮活性的削弱小。此种蓝藻的放氢对CO和氧都敏感,预先以乙炔处理时,其放氢即受抑制,而在光下以分子氢预处理则促进放氢。这类蓝藻光下放氢比暗中高,当添加光合抑制剂或氯化铵于反应系统时,其放氢便明显下降。     

Interactive effects of UV-B and Cu on photosynthesis,uptake and metabolism of nutrients in a green alga Chlorella vulgaris under simulated ozone column

This study demonstrated a general reduction in photosynthesis (carbon fixation, O(2)-evolution and photochemical electron transport chain), the uptake of NH(4)(+), NO(3)(-), urea and PO(4)(3+), and activities of nitrate reductase, urease, acid phosphatase and ATPase following UV-B and copper exposure of Chlorella vulgaris in the absence or presence of 1 and 2 ppm concentrations of a 4-inch-thick ozone layer. Though the effect of stressors used in combination was very detrimental to the above processes, selected concentrations of ozone not only counteracted the UV-B-induced inhibition of the above processes, but also stimulated O(2)-evolution and the photochemical electron transport chain. Kinetics of nutrient uptake and enzyme activities demonstrated that UV-B causes structural change(s) in the enzymes/carriers responsible for the uptake of NH(4)(+), NO(3)(-), urea and PO(4)(3+) as well as their assimilatory enzymes. Except for nitrate reductase, copper was found to compete for the binding sites of all the above enzymes. Synergistic inhibition of photosynthetic activity, nutrient (except NH(4)(+)) uptake, and enzyme activities by UV-B+Cu seems to be due to increased Cu uptake as a consequence of altered membrane permeability brought about by the peroxidation of membrane lipids in UV-B-exposed cells.