盐酸胍对大麦类囊体膜能量分配和电子传递的影响

本文以大麦叶片为实验材料,研究了盐酸胍修饰对类囊体膜能量分配及电子传递的影响。结果表明：盐酸胍处理类囊体膜,室温下Ｆ６８５荧光强度,随着盐酸胍浓度的增加而逐渐下降。盐酸胍处理导致类囊体膜在低温（７７Ｋ）下Ｆ６８５／Ｆ７８６比值下降,并随着盐酸胍浓度的增加而加剧。盐酸胍处理抑制类囊体膜以Ｈ２Ｏ为电子供体的ＤＣＩＰ光还原速度和Ｃｈｌａ诱导荧光产率,这种抑制作用可分别为加入ＰＳＩＩ的人工电子供体ＤＰＣ和     

海生植物叶绿体膜中阳离子诱导激发能分配变化的静电控制与非静电控制的研究

用Ｃａ２＋和胰酶处理大叶藻（Ｚｏｓｔｅｒａｍａｒｉｎａ）和刺松藻（Ｃｏｄｉｕｍｆｒａｇｉｌｅ）叶绿体膜,研究了它们的类囊体膜多肽组分与Ｍｇ２＋诱导Ｃｈｌａ荧光和膜表面电荷变化之间的相互关系。观察到：（１）在大叶藻的叶绿体膜中,Ｍｇ２＋诱导ＰＳ－Ⅱ荧光强度的增高与其诱导类囊体膜表面电荷密度的降低密切相关;但这种相关性的效应不存在于刺松藻的叶绿体膜中。（２）用Ｃａ２＋处理这两种叶绿体膜分别除去其类囊体膜表面的３２－３４ＫＤ和３０－３１ＫＤ多肽,对上述Ｍｇ２＋诱导的现象无明显的影响。（３）用胰酶进一步消化这些Ｃａ２＋处理过的叶绿体膜分别除去其类囊体膜表面的２６ＫＤ和２３－２４ＫＤ多肽,那么在大叶藻的叶绿体膜中,Ｍｇ２＋诱导荧光和类囊体膜表面电荷变化的相关性效应则全部消失;但在刺松藻的叶绿体膜中,Ｍｇ２＋诱导荧光增高的效应完全消失,而Ｍｇ２＋诱导膜表面电荷变化的性质则保持不变。这些实验结果不仅证明,类囊体膜表面的２６ＫＤ和２３－２４ＫＤ多肽分别为大叶藻和刺松藻叶绿体膜中阳离子诱导激发能在ＰＳ－Ⅱ和ＰＳ－Ⅰ之间分配变化的特异性作用部位;而且说明阳离子调节激发能在这两个光系统之间分配的机理,在这两种海生植物的叶绿体膜中     

阳离子诱导大叶藻叶绿体膜中激发能在PSⅡ和PSⅠ之间分配变化的机理(英文)

用Ｃａ２＋ 和胰酶处理大叶藻（Ｚｏｓｔｅｒａ ｍ ａｒｉｎａ）叶绿体膜研究了其类囊体膜多肽成分与Ｍｇ２＋ 诱导其Ｃｈｌａ荧光和类囊体膜表面电荷变化之间的相互关系,观察到：１．在正常的叶绿体膜中,Ｍｇ２＋ 诱导ＰＳⅡ荧光强度的增高与其诱导类囊体膜表面电荷密度的降低密切相关;２．用Ｃａ２＋ 处理这种叶绿体膜,除去类囊体膜表面的３２～３４ ｋＤ多肽对Ｍｇ２＋ 诱导的上述现象无影响;３．如果用胰酶消化Ｃａ２＋ 处理过的叶绿体膜,进一步除去膜表面的２６ ｋＤ多肽,Ｍｇ２＋诱导的这些现象则全部消失。这些实验结果清楚地表明,在大叶藻的叶绿体膜中,类囊体膜表面的２６ ｋＤ 多肽是阳离子诱导这两种相关现象的特异性作用部位。对阳离子调节激发能在ＰＳⅡ和ＰＳⅠ之间分配的机理进行了讨论     

海生植物叶绿体膜中阳离子诱导激发能分配变化的静电控制与非静…

用Ｃａ＾２＋和胰酶处理大叶藻和剌松藻叶绿体膜，研究了它们的类囊体膜多肽组分与Ｍｇ＾２＋诱导Ｃｈｌａ荧光和膜表面电荷变化之间的相互关系。观察到：（１）在大叶藻的叶绿体膜中，Ｍｇ＾２＋诱导ＰＳ－Ⅱ荧光强度的增高与其诱导类囊体膜表面电荷密度的降低密切相关；但这种相关性的效应不存在于剌松藻的叶绿体膜中。（２）用Ｃａ＾２＋处理这两种叶绿体膜分别除去其类囊体膜表面的３２－３４ＫＤ和３０－３１ＫＤ多肽，对上述Ｍ     

PSI颗粒中LHPC－I含量与类囊体膜状态的关系

菠菜叶绿体经低渗ＫＣｌ或１ｍｍｏｌ／ＬＥＤＴＡ溶液处理后,再用毛地黄苷法提取ＰＳＩ颗粒,前者ｃｈｌａ／ｂ高于后者。而ＥＤＴＡ－ＰＳＩ颗粒的耗氧活力极强,低温荧光发射具很强的Ｆ６８０。ＳＤＳ－凝胶电泳谱带也表明ＥＤＴＡ－ＰＳＩ颗粒具较浓的２０～２４ｋｄ谱带。凡此都说明ＥＤＴＡ－ＰＳＩ颗粒的ＬＨＰＣ～Ｉ含量远超过ＫＣＩ－ＰＳＩ颗粒的。而叶绿体经ＭｇＣｌ２、ＫＣｌ、ＥＤＴＡ溶液处理后,基粒类囊体膜垛叠的情况不同,它们的ｃｈｌａ／ｂ比值依次下降。说明ＰＳＩ颗粒中ＬＨＰＣ－Ｉ含量与提取时类囊体膜垛叠状态有关。     

PSI颗粒中LHPC—I含量与类囊体膜状态的关系

菠菜叶绿体经低渗ＫＣＬ或１ｍｍｏｌ／ＬＥＤＴＡ溶液处理后,再用毛地黄苷法提取ＰＳＩ颗粒,前者ｃｈｌ ａ／ｂ高于后者。而ＥＤＴＡ－ＰＳＩ颗粒的耗氧活力极强,低温荧光发射具很强的Ｆ６８０。ＳＤＳ－凝胶电脉谱带也表明ＥＤＴＡ－ＰＳＩ颗粒具较浓的２０－２４ｋｄ谱带。凡此都说明ＥＤＴＡ－ＰＳＩ颗粒的ＬＨＰＣ－Ｉ含量远超过ＫＣＬ－ＰＳＩ颗粒的。而叶绿体经ＭｇＣｌ２、ＫＣｌ、ＥＤＴＡ溶液处理后,基粒类囊体膜垛叠     

两价阳离子在类囊体膜上H~＋－ATP酶活化中的作用

在高盐介质中用ＥＤＴＡ去除叶绿体内源游离Ｍｇ２＋后制备的类囊体具有与正常类囊体相似的△ｐＨ幅度和光合磷酸化反应速率,即膜上ＣＦ０与ＣＦ１仍处于正常的耦联状态。以此为材料研究不同两价阳离子（Ｍ２＋）在ＡＴＰ酶活化及催化反应中的作用,结果表明：（１）Ｍｇ２＋Ｃａ２＋Ｍ２＋Ｚｎ２＋Ｃｏ２＋Ｂａ２＋分别对光下ＡＴｈ形成与水解的效应大小与它们的离子半径有一定关系。（２）这些Ｍ２＋以不同方式不同程度地抑制了Ｍｇ２＋－ＡＴＰ酶的合成或水解ＡＴＰ的活性。（３）低浓度Ｍ２＋的电荷屏蔽效应可稳定酶在光下的活化构象,这有利于暗中进行的需Ｍｇ２＋的催化ＡＴＰ合成与水解反应。     

Mg~（2＋）促进线粒体H~＋－ATP酶的重建机理──H~＋－ATP酶复合体亚基水平的研究

用专一性标记蛋白质巯基的荧光探剂ａｃｒｙｌｏｄａｎ测定含Ｍｇ＾２＋的Ｆ０－ＡＴＰ酶或Ｆ０－ＯＳＣＰ－Ｆ１－ＡＴＰ酶的脂酶体的构象与无Ｍｇ＾２＋者明显不同,前者的蛋白质的－ＳＨ基团处于疏水性更强的微环境中;在有Ｍｇ＾２＋和ＯＳＣＰ同时存在下重建的Ｆ０－Ｆ１－ＡＴＰ酶脂酶体较无ＯＳＣＰ者表现更高的水解活力或膜电位,表明ＯＳＣＰ增强Ｍｇ＾２＋的促进作用,这进一步提示Ｍｇ＾２＋通过改变膜脂的物理状态促进线     

Na2SO3和NaHCO3对叶绿体CF1—ATPase活力作用的机制

Ｎａ２ＳＯ３对热－ＤＴＴ活化的游离ＣＦ１及类囊体膜上ＣＦ１－ＡＴＰａｓｅ活力均有显著的促进作用,ＮａＨＣＯ３亦有明显的促进作用,Ｎａ２ＳＯ３和ＮａＨＣＯ３的促进作用与它们解除Ｍｇ＾２＋的抑制作用有关,从ＮａＨＣＯ３和Ｎａ２ＳＯ３及它们与Ｍｇ＾２＋之间的竞争性关系。表明三者是结合在酶的同一部位上。Ｎａ２ＳＯ３可明显降低热－ＤＴＴ活化的游离ＣＦ－ＡＴＰａｓｅ催化反应的活化能,这可能与促进产物ＡＤＰ的翻     

Mg~（2＋）促进线粒体H~＋－ATP酶的重建机理──H~＋－ATP酶复合体亚基水平的研究

用专一性标记蛋白质巯基（－ＳＨ）的荧光探剂ａｃｒｙｌｏｄａｎ测定含Ｍｇ２＋的Ｆ０－ＡＴＰ酶或Ｆ０－ＯＳＣＰ－Ｆ１－ＡＴＰ酶的脂酶体的构象与无Ｍｇ２＋者明显不同,前者的蛋白质的－ＳＨ基团处于疏水性更强的微环境中;在有Ｍｇ２＋和ＯＳＣＰ同时存在下重建的Ｆ０－Ｆ１－ＡＴＰ酶脂酶体较无ＯＳＣＰ者表现更高的水解活力或膜电位,表明ＯＳＣＰ增强Ｍｇ２＋的促进作用,这进一步提示Ｍｇ２＋通过改变膜脂的物理状态促进线粒体Ｈ＋－ＡＴＰ酶重建的间接作用。这些实验结果,从线粒体Ｈ＋－ＡＴＰ酶复合体的亚基水平的相关性上,对于我们提出的Ｍｇ２＋通过改变膜脂的物理状态使之具有合适的流动性,诱导嵌入脂双层的Ｈ＋－ＡＴＰ酶复合体的Ｆ０的构象发生变化并传递至复合体的催化中心Ｆ１,从而使重建Ｆ１－Ｆ０－ＡＴＰ酶具有较适合的蛋白构象,表现较高的重建酶活性的假设提供了直接的实验证据,精确地阐明了Ｍｇ２＋促进线粒体Ｆ０－Ｆ１－ＡＴＰ酶重建作用的分子机理。     

Mechanism of Change in the Cation-Induced Excitation Energy Distribution Between PSⅡ and PS Ⅰ in the Chloroplast Membrane from Zostera marina

The interrelations between thylakoid polypeptide components and Mg2+-induced Chl a fluorescence and thylakoid surface charge changes were investigated in Zostera marina chloroplasts treated with Ca2+ and trypsin. It was observed that: 1. The increase of Mg2+- induced PS Ⅱ fluorescence intensity was closely related to the decrease of Mg2+-induced surface charge density of the thylakoid membrane in the normal chloroplast; 2. Removal of the 32～34 kD polypeptides of the thylakoid surface by Ca2+ extraction of the chloroplast did not affect the Mg2+-induced phenomena; 3. If the Ca2+-treated chloroplast was further digested by trypsin to remove the 26 kD polypeptide of the membrane surface, the Mg2+-induced phenomena disappeared completely. These results clearly indicated that the 26 kD polypeptide of thylakoid surface is the specific acting site of the cation that induced these two correlated phenomena in the chloroplast from Zostera marina. The mechanism on the regulating effect of the cation on excitation energy distribution between PS Ⅱ and PS Ⅰ was discussed.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.     

The Regulation and Distribution of LHC-I and LHCP2 between the Photosystem I and Photosystem II

Evidences were provided in this paper that the relative distribution of chl-protein complexes of PSⅠ and PSⅡ could be regulated by Mg2+. addition of Mg2+ led to decrease in the amount of chl-protein complexes of PSⅠ and increase in the amount of chl-protein in complexes of PSⅡ. There was no effect of Mg2+ on the spectral property of LHCP1, but the addition of Mg2+ could change the spectral property of LHCP2 so that it became similar to that of the LHC-Ⅰ. CPIa2 was a complex of reaction centre of PSⅠ and LHC-I. LHC-I might be contacted specially with LHCP2 in chloroplast membranes. Addition of Mg2+ probably cansed the motion of LHC-I from PSⅠ to PSⅡ and became more closely connected with LHCP2. The relative amount of CPIa2, CPIa1, LHCP1 and LHCP2 in chloroplast membranes could be regulated by different light intensity. There were more CPIa2, LHCP1 and less LHCP2 in chloroplast membranes from the shade plant Malaxis monophyllos and sunflower grown under weak light, both of them lacked equally CPIa1. There were less CPIa2, LHCP1 and more LHCP2 in the sun plant spinach and sunflower grown under strong light, and they possessed equally CPIa1 chl-protein complexes. It is suggested that LHCP1 and LHCP2 are different light-harvesting Chl-protein complexes. The LHC-I and LHCP2 are mobile light-harvesting chl-protein complexes and shuttle back and forth between PSⅠ and PSⅡ They play an important role in the regulation and distribution of excitation energy between the two photosystems.     

盐酸胍对毕氏海蓬子类囊体膜蛋白光谱特征的影响

研究了盐酸胍（GuHCl）处理对毕氏海蓬子类囊体膜蛋白亚基和光谱特征的影响。结果显示,随着GuHCl处理浓度的增高,类囊体膜蛋白的吸收光谱和荧光发射光谱明显下降,峰位发生蓝移。这表明GuHCl处理下,类囊体膜蛋白色素微环境发生明显变化,色素蛋白结构遭到破坏;较低浓度2mmol.L-1GuHCl处理下,随着GuHCl处理时间的延长,类囊体膜蛋白的吸收光谱和荧光发射光谱也呈下降趋势,但与GuHCl浓度梯度处理比,下降程度略缓,峰位也基本没有变化。这说明类囊体膜在2mmol.L-1GuHCl不同处理时间下表现出的耐受性比在GuHCl浓度梯度处理条件强。     

Dephosphorylation of the thylakoid membrane light-harvesting complex-II by a stromal protein phosphatase

Light-harvesting complex-II (LHC-II) phosphatase activity has generally been examined in the intact thylakoid membrane. A recent report of peptide-phosphatase activity associated with the chloroplast stromal fraction (Hammer, M.F. et al. (1995) Photosynth Res 44: 107–115) has led to the question of whether this activity is capable of dephosphorylating membrane-bound LHC-II. To this end, heat-treated thylakoid membranes were examined as a potential LHC-II phosphatase substrate. Following incubation of the thylakoid membrane at 60°C for 15 min, the endogenous protein phosphatase and kinase activities were almost eliminated. Heat-inactivated phosphomembranes exhibited minimal dephosphorylation of the light harvesting complex-II. Peptide-phosphatase activities isolated from the thylakoid and stromal fraction were able to dephosphorylate LHC-II in heat-inactivated phosphomembranes. The stromal phosphatase showed highest activity against LHC-II at pH 9. Dephosphorylation of the LHC-II by the stromal enzyme was not inhibited by molybdate, vanadate or tungstate ions, but was partially inhibited by EDTA and a synthetic phosphopeptide mimicking the LHC-II phosphorylation site. Thus, the previously identified stromal phosphatase does appear capable of dephosphorylating authentic LHC-II in vivo.Abbreviations CPP chymotryptic phosphopeptides - LHC-II light-harvesting complex of Photosystem II - MP protein phosphatase fractionated from the thylakoid membrane - P_2Thr synthetic phosphopeptide MRK-SAT(p)TKKVW - SP protein phosphatase fractionated from the stromal compartment     

Changes of Freeze-Fracture Ultrastructure and Light Harvesting Chlorophyll Protein Complex in Thylakoid Membranes During Ontogeny of Maize

Freeze-fracture electron microscopy enables us to observe and count the freeze-fracture particles which correspond to the different functional components of thylakoid membranes. The present paper reports the observation on freeze-fracture ultrastructure of thylakoid membranes and the analysis of proteins by the SDS-polyacrylamide gel electrophoresis within the membranes from differenly located leaves of maize. In the past, we found that the leaies subtending the ear of maize had a much higher chlorophyll content, a lower chlorophyll a/b ratio and more staking thylakoid membranes and provided the photosynthetic energy used to fill the maize seeds more than that of other leaves. Recently, we have further found that the particle densities of all four faces of thylakoid membranes from the ear leaf were the highest, than those, successively, from the terminal leaf, and the fifth leaf (from the base of the plant). The particle densities on all four fracture faces of thylakoid membranes isolated from the ear leaves of maize were significantly higher than those from the terminal leaves with the increases of 19% in EFs, 28% in PFs and 20% in PFu. Increases in particle densities on the PFs, EFs and PFu faces result in increased densities of LHCP II, PSⅡ and PSI reactions centres, respectively. It is significant that this supramolecular architecture of the ear leaves is consistent with our analytical results of the SDS-polyacrylamide gel electrophoresis within the membranes (a detailed report in another paper). The contents of major polypeptides of 21 kD (LHCP Ⅰ) and 25 kD (LHCP Ⅱ) in thylakoid membranes from the ear leaves were more than those from the terminal leaves. The characteristics of both supramolecular architecture and polypeptide components are in favour of absorbing, transferring, distributing and conversing light energy in the course of photosynthesis of the ear leaves in maize.     

Protein phosphorylation and Mg2+ influence light harvesting and electron transport in chloroplast thylakoid membrane material containing only the chlorophyll-a/b-binding light-harvesting complex of photosystem II and photosystem I.

A material containing only photosystem I (PSI) and the chlorophyll-a/b-binding light-harvesting complex of PSII (LHC-II) has been isolated from the chloroplast thylakoid membrane by solubilization with Triton X-100. Fluorescence spectroscopy shows that, within the material, LHC-II is coupled to PSI for excitation-energy transfer and that this coupling is decreased by the presence of Mg2+, which also decreased PSI electron transport specifically at limiting light intensity. Inclusion of phosphorylated LHC-II within the material did not alter its structure, but gave decreased energy transfer to PSI and inhibition of electron transport which was independent of light intensity, implying effects of phosphorylation on both light harvesting and directly on electron transport. Inclusion of Mg2+ within the phosphorylated material gave decreased energy transfer, but slightly increased PSI electron transport. A cation-induced direct promotion of PSI electron transport was also observed in isolated PSI particles. The PSI/LHC-II material represents a model system for examining protein interactions during light-state adaptations and the possibility that LHC-II can contribute to the antenna of PSI in light state 2 in vivo is discussed.     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll a/b-protein complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl a/b-protein complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.