两种电泳系统分离裙带菜色素-蛋白复合物的比较

裙带菜的类囊体膜经过去污剂癸基-N-甲基匍萄糖胺增溶,采用SDS-PAGE分离技术,在Tris-Gly和Tris-硼酸两种电泳系统中分离其色素-蛋白质复合物,并比较其复合物的光谱特性。结果表明:采用Tris-Gly电泳分离系统从裙带菜中分离到8种色食-蛋白质复合物,分别是CP Ia、CPI、LHC₁、CPa、LHC₂、LHC₃、LHC₄和LHC₅。在Tris-硼酸电泳分离系统中共分离到5种色素-蛋白质复合物,分别是CPI、CPa、LHC₁、LHC₂、LHC₃。吸收光谱和荧光光谱的测定结果表明,两种电泳系统中分离的相对应条带的光谱特性基本相近。     

绿藻假根羽藻色素-蛋白复合物的分离及其特性研究

采用聚丙烯酰胺凝胶电泳(PAGE)和蔗糖密度梯度超速离心方法分离了假根羽藻(Bryopsis corticulans)的色素-蛋白复合物,并对其特性进行分析。结果表明:采用PAGE分离得到7条色素-蛋白复合物带,分别是CPⅠa1、CPⅠa2、CPⅠ、LHCP₁、LHCP₂、CPa、LHCP₃₊₃,和2条游离色素(free pigment,FP)FCa、FC。用改进的不连续蔗糖密度梯度离心法分离到五条带。区带Ⅰ是FP;区带Ⅱ主要是小分子量的PSⅡ捕光复合物LHCP₃₊₃;区带Ⅲ以PSⅡ捕光复合物的聚集体LHCP1为主,区带Ⅱ和Ⅲ的吸收光谱中除了Chla外,还含有大量的Chlb和管藻黄素,是管藻黄素-Chla/b-蛋白质复合物;区带Ⅳ在PAGE中只显示一条带,光谱中有Chlb吸收肩峰,含有66和56kDa两种多肽,是较小的PSⅠ复合物CPⅠa。     

褐藻裙带菜中PSⅡ复合物及其亚复合物的分离和特性研究

用不连续梯度蔗糖密度超离心,从经Triton X-100增溶的褐藻裙带菜类囊体膜中分离到3种色素蛋白复合物条带,分别是捕光复合物、具有光氧化活性的PSⅡ复合物颗粒（区带Ⅱ）以及PSⅠ（区带Ⅲ）。PSⅡ颗粒经毛地黄皂苷增溶后,再次超离心分离得到3条PSⅡ的亚复合物条带。吸收和荧光激发谱显示其中的区带Ⅱ-1为墨角藻黄素-Chl a／c-蛋白复合物,区带Ⅱ-2为Chl a／c-蛋白复合物,两者都只含20kDa多肽;而鲜绿色的区带Ⅱ-3为不含捕光复合物的活性PSⅡ核心。     

褐藻裙带菜中PSII复合物及其亚复合物的分离和特性研究

用不连续梯度蔗糖密度超离心,从经TritonX-100增溶的褐藻裙带菜类囊体膜中分离到3种色素蛋白复合物条带,分别是捕光复合物、具有光氧化活性的PSII复合物颗粒(区带II)以及PSI(区带III)。PSII颗粒经毛地黄皂苷增溶后,再次超离心分离得到3条PSII的亚复合物条带。吸收和荧光激发谱显示其中的区带II-1为墨角藻黄素-Chla/c-蛋白复合物,区带II-2为Chla/c-蛋白复合物,两者都只含20kDa多肽;而鲜绿色的区带II-3为不含捕光复合物的活性PSII核心。     

海洋管藻目绿藻刺松藻光系统Ⅰ复合物的分离

采用Triton X-100蔗糖密度梯度离心法,从管藻目绿藻刺松藻中分离到三种不同形式的光系统Ⅰ（PSⅠ）复合物.区带Ⅲ富含PSⅠ核心复合物（CCⅠ）,叶绿素（Chl）a/b＞20,在温和的聚丙烯酰胺凝胶电泳（PAGE）中只显示一条PSⅠ中心复合物CPⅠ条带.区带Ⅳ和Ⅴ在436和674 nm、467和650 nm以及540 nm的吸收表明,含有Chl a、b及管藻黄素和管藻素,Chl a/b比值分别为3.23和2.4.经PAGE检测,有CPⅠ和CPⅠa两种PSⅠ色素蛋白复合物带,因此区带Ⅳ和Ⅴ是由CCⅠ和含量不等的捕光复合物LHCⅠ构成的PSⅠ颗粒.区带Ⅲ只有66和56 ku两种核心多肽;区带Ⅳ和Ⅴ除了66、56 ku多肽以外,还有4种分子质量为25,26,26.2和27.5 ku的LHCⅠ多肽.室温荧光光谱显示,分离物中的各种光合色素之间保持着良好的能量传递关系,由Chl b及管藻黄素和管藻素吸收的能量都可以传递给Chl a.     

从蓝藻Anabaena cylindrica囊状体膜分离的叶绿素蛋白复合体

当蓝藻的囊状体膜在SDS与叶绿素之比为10:1的条件下增溶后,经不连续的SDS-聚丙烯酰胺凝胶电泳分离出六条含叶绿素的带。按照电泳迁移率增加的顺序,以及吸收光谱和荧光光谱的鉴定结果,自上而下分别命名为CP1a,CP1b,CP1c,CP1,CPa和FC。 CP1a,CP1b,CP1c和CP1四种复合体在蓝区和红区的吸收峰分别位于435 nm和675 nm处。该四种复合体在77°K的荧光发射峰位于726～728 nm。铁氰化钾-抗坏血酸氧化还原差异光谱证明这四种复合体都含有 P 700, 说明它们属于光系统Ⅰ反应中心复合体。低温荧光激发光谱表明这些复合体在625～626 nm,677 nm,690～692 nm和712～714 nm处有四个共同的荧光激发峰或肩。根据其E677/E714的比值,可将它们分为CP1a,CP1b和CP1c,CP1两种类型。它们之间的差异在于这两类复合体之间不同状态的色素比例明显不同。 第五种叶绿素蛋白复合体CPa在蓝区的吸收峰位于435nm处,在红区的吸收峰位于672nm处,CPa在77°K的荧光发射峰位于686 nm处,另外在690～696nm范围内还有一个较弱的肩。它属于光系统Ⅱ反应中心复合体。它仅存在于营养胞中。 异形胞中只有光系统Ⅰ反应中心复合体。     

裙带菜PSⅠ复合物的荧光特异性

以褐藻裙带菜（Undaria pinnatifida）为实验材料,采用蔗糖密度梯度超速离心的方法,去污剂SDS为增溶剂（SDS:Chl＝20:1,4℃增溶20 min）,蔗糖密度梯度为60%、50%、40%、30%、20%、15%和10%,分离制备光系统Ⅰ(PSⅠ)复合物。结果表明, 40% 蔗糖层带所含色素蛋白复合物是PSⅠ复合物。利用红藻作参照对比,光谱结果表明从裙带菜中得到的PSⅠ复合物没有730 nm的荧光峰。分析认为这是所有褐藻包括裙带菜PSⅠ复合物的荧光特异性。     

裙带菜PSⅠ复合物的荧光特异性

以褐藻裙带菜(Undaria pinnatifida)为实验材料,采用蔗糖密度梯度超速离心的方法,去污剂SDS为增溶剂(SDS:Chi=20:1,4℃增溶20 min),蔗糖密度梯度为60%、50%、40%、30%、20%、15%和10%,分离制备光系统Ⅰ(PSⅠ)复合物.结果表明,40%蔗糖层带所含色素蛋白复合物是PS I复合物.利用红藻作参照对比,光谱结果表明从裙带菜中得到的PSⅠ复合物没有730 nm的荧光峰.分析认为这是所有褐藻包括裙带菜PSⅠ复合物的荧光特异性.     

裙带菜与菠菜的色素-蛋白质复合体的比较研究

裙带菜ＰＳＩ复合体的７７Ｋ荧光发射光谱与菠菜的明显不同,缺少７３０ｎｍ长波荧光峰,位于７１５ｎｍ处,其捕光色素－蛋白质复合体有墨角藻黄素－叶绿素ａ／ｃ－蛋白质复合体和叶绿素ａ／ｃ－蛋白质复合体两种,菠菜只有一种叶绿素ａ／ｂ－蛋白质复合体。     

固氮蓝藻Anabaena sp．7120的叶绿素蛋白复合体的分离和光谱特性的研究

用光合膜片增溶和SDS-聚丙烯酰胺凝胶电泳方法,从固氮蓝藻Anabaena sp．7120分离到7条色素带。迁移率较慢的五条叶绿素蛋白复合体带,具有相同的吸收光谱和室温荧光光谱特性。它们的红区最大吸收峰在676nm;蓝区最大吸收峰在438nm。它们的室温荧光发射最高峰在672-673nm;在710,732和740nm都有小峰。这些是CPI叶绿素所特有的。我们认为这5条带都是属于光系统Ⅰ的叶绿素蛋白复合体。另一条迁移率稍快的叶绿素蛋白复合体带为CPⅡ。它的红区最大吸收峰在672nm;蓝区最大吸收峰在436nm。与CPⅠ带相比,两个峰均向短波端偏移。它们的室温荧光发射最高峰在675nm,没有CPⅠ所特有的小峰。这些性质说明此带和CPⅠ带不同,而是和光系统Ⅱ反应中心相关的一个复合体。迁移率最快的带是游离色素带。     

The Chlorophyll-Protein Complexes Resolved from Ultrasonically Broken Thylakoid Memb-ranes of Blue-Green Algae

When the thylakoid membranes of blue-green algae were broken by ultrasonic vibrations and subjected to polyacrylamide gel electrophoresis at 4℃, six green zones were resolved. They were designated as CPIa, CPlb, CPI; CPal, CPa2, and FC. The absorption spectrum of CPI had a red maximum at 674 nm and a peak in the blue at 435 nm. It was identified as PS chlorophyll a-protein Complex, but was contaminated with minor PSⅡ which was implied by the appearance of fluorescence emission peak at 680 nm besides the main one at 725 nm at 77 K. The spectral properties of CPIa and CPlb were similar to that of CPl. The absorption spectra of CPa1 and CPa2 were similar, both having red maxima at 667 nm and peaks in the blue at 431.5 nm. Their fluorescence emission had the same peaks at 684 nm at 77 K indicating that they belonged to PSⅡ. It was recognized that CPal of 47 kD is the reaction center complex of photosystem Ⅱ and CPa2 of 40 kD is the internal antenna complex of photosystem Ⅱ. The spectral characteristics of the chlorophyll-protein complexes resolved by ultrasonic method were similar to those of the same complexes resolved by SDS solubilization, except the absorbance positions of CPa1 and CPa2 in the blue peak and the red one which shifted to blue about 3–5 nm. It was calculated that in thylakoid membranes of blue-green algae 40.93% chlorophyll was in PSⅠ, while 38.78% of chlorophyll in PSⅡ. The difference of chlorophyll contents between PSⅠ and PSⅡ was only 2.15%. Concerning the fact that minor PSⅡ compound remained in the part of PSⅠ zones, it might be concluded that the distribution of chlorophyll between PSⅠ and PSⅡ in blue-green algae was equal. This result was in agreement with the hypothesis that PSⅠ and PSⅡ operates in series in photosynthetic electron transport.     

A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Chlorophyll-Protein Complexes of Blue-Green Algae Isolated by a New Gel Electrophoresis System with High Resolving Power

At least 13 chlorophyll bands from the thylakoid membranes of blue-green algae could be clearly resolved by SDS-PAGE employing a new improved procedure. They were designated as CPIa, CPIb, CPIc, CPId, CPIe, CPIf, CPIg, CHIh, CPal, CPa2, CPa3, CPa4 and FC. 8 chlorophyll-protein complexes, CPIa-CPIh, had the same absorption spectrum at 676 nm in the red and 436 nm in the blue region. They belonged to the chlorophyll-protein complexes of PS Ⅰ. 4 chlorophyll-protein complexes, CPal-CPa4, had a red absorption peak at 670­672 nm and a blue one at 436 nm. Their fluorescence emission peak at 77K was at 685 nm. They were chlorophyll-protein complexes of PS Ⅱ.     

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg²⁺. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg²⁺ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg²⁺ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. V-separation and characterization of pigment-protein complexes of the differentiated thylakoids

Low temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis following mild solubilization of Euglena thylakoid components allowed to resolve, in addition to the main CP1, CPa and LHCP chlorophyll-protein complexes, the additional CP1a and LHCP green bands. A carotenoid enriched band CPc can be separated from CPa using high acrylamide concentration. Pigment and polypeptide composition of these complexes were analyzed by absorption and fluorescence measurements and two dimensional gel electrophoresis. Spectral properties of CP1 and CP1a indicate an heterogenous organization of chlorophyll and the presence of significant amount of chlorophyll b in these complexes. They both contain a major 68 kilodalton polypeptide associated with three minor low molecular weight polypeptides in CP1a. CPa and CPc exhibit a characteristic fluorescence emission at 687 nm and they each contain one polypeptide of 54 and 41 Kda respectively. LHCP and LHCP are less abundant than in higher plant thylakoids and they contain a lower proportion of chl b (chl a: chl b=3). They include two polypeptides of 26 and 29 Kda.Abbreviations chl chlorophyll - SDS Sodium Dodecyl Sulfate - EDTA Ethylene Diamine Tetraacetic Acid - DTT Dithiothreitol     

Origin of the F685 and F695 fluorescence in Photosystem II

The emission spectra of CP47-RC and core complexes of Photosystem II (PS II) were measured at different temperatures and excitation wavelengths in order to establish the origin of the emission and the role of the core antenna in the energy transfer and charge separation processes in PS II. Both types of particles reveal strong dependences of spectral shape and yield on temperature. The results indicate that the well-known F-695 emission at 77 K arises from excitations that are trapped on a red-absorbing CP47 chlorophyll, whereas the F-685 nm emission at 77 K arises from excitations that are transferred slowly from 683 nm states in CP47 and CP43 to the RC, where they are trapped by charge separation. We conclude that F-695 at 77 K originates from the low-energy part of the inhomogeneous distribution of the 690 nm absorbing chlorophyll of CP47, while at 4 K the fluorescence originates from the complete distribution of the 690 nm chlorophyll of CP47 and from the low-energy part of the inhomogeneous distribution of one or more CP43 chlorophylls.     

Characterization of a 31 kDa polypeptide that accumulates in the light-harvesting apparatus of maize leaves during chilling

A 31 kDa polypeptide that accumulates in the thylakoids when maize leaves are chilled to 5°C in the light is characterized using monoclonal antibodies and analyses of chlorophyll-protein complexes. This polypeptide reacted with a monoclonal antibody, MLH2, that was specific for the 28 kDa polypeptide of the light-harvesting complex (LHCII) of pea leaves. On chilling leaves the appearance of a chlorophyll-protein complex having an apparent molecular weight of 31 kDa coincided with the appearance of a 31 kDa polypeptide and a decrease in the 29 kDa chlorophyll-protein, CP29. Returning the leaves to 25°C for 1 h produced a loss of both the 31 kDa chlorophyll-protein and 31 kDa polypeptide from the thylakoids, and an increase in the amount of CP29. Breakdown of the 31 kDa polypeptide in vitro was Mg²⁺-dependent and inhibited by EDTA and transition metal ions. It is suggested that the 31 kDa polypeptide may be a precursor of the apoprotein of CP29 and can bind chlorophyll. The appearance of the 31 kDa polypeptide correlated with a marked change in the 77 K fluorescence emission spectra of isolated LHCII particles, which did not revert with the disappearance of the 31 kDa on returning the leaves to 25°C for 1 h. The physiological significance of this spectral perturbation is discussed.