藻胆体核亚基ApcD定点突变及体内重组功能研究

在对Anabaena sp.PCC7120藻胆体核亚基ApcD结合色素PCB的体内重组中,发现色素蛋白在提纯前后最大吸收峰和荧光峰发生了红移,从提纯前的605nm及633nm变为提纯后的650nm及665nm.为了研究该现象的原因,构建了ApcD的8个突变体,重组结果显示:突变体ApcD(Y88I)色素蛋白在提纯后的吸收光谱和荧光光谱较提纯前均多出一个峰,分别为668nm,690nm;ApcD(W59Q)、ApcD(Y73A)、ApcD(W87E)色素蛋白在提纯前后的吸收光谱和荧光光谱一致;ApcD(M126S)、ApcD(Y116S)、ApcD(M160T)色素蛋白在提纯前后的吸收光谱一致,而提纯后的荧光峰位置较提纯前分别红移了5nm、7nm和10nm;ApcD(M115I)色素蛋白在提纯前后的吸收光谱和荧光光谱均发生了红移,从提纯前的605nm和633nm变为提纯后的638nm和655nm.这些色素蛋白在酸性尿素溶液变性条件下的最大吸收峰始终在662nm,表明辅基色素仍然是藻蓝胆素;在对PCB-ApcD、PCB-ApcD(Y116S)及PCB-ApcD(M160T)的圆二色谱分析发现,该两个氨基酸的突变均...     

鱼腥藻PCC7120核膜连接蛋白ApcE体内重组研究

为了研究鱼腥藻PCC7120核-膜连接蛋白ApcE（1-240）脱辅基蛋白与藻蓝胆素的连接机制,通过体内重组方式得到色素蛋白PCB-ApeE（1-240）。吸收光谱、荧光光谱分析表明,核-膜连接蛋白ApcE（1-240）与藻蓝胆素进行了正确的体内重组。ApeE（1-240）脱辅基蛋白可与藻蓝胆素体内自催化共价连接,获得的色素蛋白溶于含有4mol／L尿素的磷酸钾缓冲体系中,并具有最大吸收峰（λmax=660nm）和荧光发射峰（λmax=668nm）。     

近红外与荧光光谱法检测血糖试验研究

采用荧光光谱法检测血糖。结果表明,检测血清的最佳激发波长为340 nm,血清中葡萄糖的发射峰位置为470 nm。随着血清中葡萄糖浓度的增加,荧光光谱图中荧光峰处荧光强度,面积积分强度整体呈上升趋势,荧光峰的半峰全宽整体呈现下降趋势。利用近红外光谱法检测血糖,血清可分辨的光谱区域位于5 600~6 060 cm-1范围内,不同血糖浓度的吸收峰均位于5 768 cm-1处。随着血糖浓度的增大,其吸收峰处的吸光度逐渐增大,面积积分强度也呈上升趋势。分析二者的优缺点,荧光分析法受外界因素的干扰更小,精确度更高,光谱参数与血糖浓度之间的规律更明显,在有创检测方面具有更明显的优势,而近红外光谱法检测血糖则在无创血糖检测方面具有更大的潜力。     

Nd:YAP激光辐照对雨生红球藻生理效应的荧光分析

目的：Nd：YAP激光辐照对雨生红球藻生理效应的荧光分析j方法：利用Nd：YAG激光（功率10W,辐照剂量为15s、35s、55s）辐照雨生红球藻,在对辐照细胞进行生长测定以及色素吸收光谱分析的基础上,进一步利用激光共聚焦扫描显微镜（LSCM）,在波长488nm的Ar^＋激发光条件下,对细胞的自体荧光以及吖啶橙染色的细胞核荧光物质进行定位定量分析,获得细胞自发荧光光谱和细胞核荧光物质的荧光光谱。结果：①低剂量的Nd：YAG激光辐照（15s左右）对藻细胞有促长作用,生长速率提高20．3％,色素吸收峰的峰值提高25．3％。较高剂量的Nd：YAG激光辐照（35s、55s）对藻细胞生长有抑制作用,并有明显的致死、致突效应。②对自体荧光的分析结果表明,与对照组相比,低剂量激光辐照组的细胞,在荧光光谱的峰型及峰值上变化不大,在682nm处均有较强的荧光发射,而高剂量激光辐照组的细胞多无明显的荧光发射。③对细胞核荧光物质的分析,雨生红球藻细胞在530nm（DNA）和640nm（RNA）处均有荧光发射峰。与对照组相比,低剂量激光辐照组的DNA荧光发射有所增强,但RNA的荧光发射则有所减弱;高剂量激光辐照组的核物质荧光发射谱,在峰型上与对照组存在差异,荧光强度也明显下降。结论：低剂量的Nd：YAP激光辐照对细胞核的DNA合成与复制有一定的刺激作用,可促进光合色素的合成并提高其对光能的吸收效率,从而增强光合活性,促进细胞的增殖与生长;高剂量激光辐照则对细胞的DNA有损伤作用,是致死率上升并发生突变的可能原因。     

Fe3+对雨生红球藻(Haematococcus pluvialis)生长及荧光特性的影响

为探求适宜雨生红球藻CG-06株生长的Fe³⁺浓度和Fe³⁺对藻细胞荧光特性的影响,以BBM为基础培养基,选用EDTA-FeNa(Ⅲ)为Fe³⁺源,设置0、1.79、8.95、17.9、35.8、71.6μmol·L^-16种Fe³⁺浓度梯度,实验测定藻的生长并分析不同Fe³⁺对藻细胞叶绿素荧光和77 K低温荧光等的影响。结果表明:适合雨生红球藻CG-06株生长的Fe³⁺浓度为8.95μmol·L^-1,Fe³⁺浓度较高时,雨生红球藻的生长受到抑制,Fe³⁺浓度低于1·79μmol.L^-1将产生低铁限制。W alz-PAM测定数据显示,在铁不足或高铁抑制条件下,雨生红球藻光系统Ⅱ活性明显下降,开放态的反应中心数目减少,光合作用受到抑制。77 K低温荧光光谱显示,在铁不足或高铁抑制条件下,710 nm荧光峰降低,684 nm和694 nm荧光峰相对增强,说明能量在两个光系统分配上发生变化,能量更多的分配给光系统Ⅱ,限制了光系统Ⅰ的活性。在高铁抑制条件下,CP₄₇蛋白荧光峰降低,CP₄₃蛋白荧光峰增强,推测存在D₁蛋白降解。     

热诱导PS核心复合物荧光动力学研究

运用UV-3101PC型紫外-可见-红外分光光度计及皮秒时间分辨荧光光谱技术对20℃、42℃、48℃3个温度下,PS 核心复合物的吸收光谱和时间分辨荧光光谱的变化进行分析,结果发现: 1 在PS 核心复合物中至少存在以下几种具有特征吸收峰的chla分子,CP43:chla660/661 chlaa:a代表吸收峰 、chla669、chla671、chla682/683;CP47:chla660/661、chla669、chla671/672、chla688、chla680;RC:chla680、chla670、chla684、chla673/674、chla682/683、chla660.吸收峰波长随测量温度等条件的不同而略有变化. 2 荧光发射谱组分的峰值随温度的升高而发生明显的蓝移,这是由于热诱导改变了蛋白质的结构,从而使生色团间的距离和 或 方位受到了影响,而chla分子的结构并未发生变化,因此导致chla分子间激子相互作用被破坏,从而产生了发射峰蓝移的现象. 3 20℃、42℃时核心天线向反应中心的能量传递是高效有序的. 4 反应中心中与蛋白质存在不同结合的chla分子,以及核心天线中吸收不同波段光的chla分子与蛋白质结合方式随温度变化存在不同反应.     

YHPD及其分离组份在溶液中和CHO细胞内的吸收和荧光特征

本文报道了YHPD及其用HPLC方法分离得到的四种主要组份在溶液中和CHO细胞内的吸收和荧光特性.YHPD分离组份d在稀溶液状态(0.5μg/ml)与其它三种组份不同,表现出较稳定的聚集态性质,YHPD及组份摄入细胞后,荧光光谱和Soret吸收峰明显红移,荧光量子产额增高.同时看到YHPD的吸收和荧光特性受环境中H~ 浓度的影响     

几种绿藻、褐藻和红藻的吸收光谱的比较研究

1.用乳白玻璃法测定了我国黄海常见的属于绿藻门、褐藻门和红藻门的十种海藻的吸收光谱。讨论了这三门海藻的吸收光谱的特点。通过对吸收光谱及红藻、褐藻相对于绿藻的差异光谱的分析,讨论了各种色素在活体中的吸收峰和吸收带的位置。2.根据褐藻与绿藻的吸收光谱的差异及差异光谱的比较分析,认为绿藻吸收光谱中位于485(或480)毫微米的吸收峰是叶绿素 b 的蓝光吸收峰。叶绿素 b 的红光吸收峰位于655毫微米附近。岩藻黄素的吸收峰位于525毫微米附近。根据红藻吸收光谱及其同绿藻的差异光谱中的红光吸收峰和负峰的特点,初步认为红藻中的叶绿素 d 的红光吸收峰比叶绿素 a 的红光吸收峰位于较长波长处。3.对几种红藻的吸收光谱进行了比较。观察到原始红藻纲的条斑紫菜(Porphyra yezoen-sis)的藻胆素与真红藻纲的藻类有显著的差异。这种差异及条斑紫菜的藻胆素的含量和组成上的易变性值得进一步研究。     

水葫芦及菠菜光合作用原初光反应的超快光谱研究

采用相同的分离技术,从水葫芦(Eichhornia crassipes(Mart)Solms.)和菠菜(Spinacia oleracea L.)叶片中提取叶绿体.利用吸收光谱和低温荧光光谱及皮秒荧光单光子计数技术对它们的光谱性质和光系统Ⅱ荧光寿命进行了研究.这两种叶绿体吸收光谱相似,暗示着它们都能高效吸收不同波长的光子.低温荧光光谱显示,水葫芦叶绿体两个光系统之间激发能分配平衡状态差,表明不利于该植物叶绿体高效利用吸收的光子能.采用三指数动力学模型对测定的光系统Ⅱ荧光衰减曲线拟合,水葫芦叶绿体光系统Ⅱ荧光衰减寿命分别是:138,521和1 494 ps;菠菜叶绿体荧光寿命分别是:197,465和1 459ps.并且归属了荧光组分,慢速度荧光衰减是由叶绿素堆积造成的,中等速度荧光衰减源于PSⅡ反应中心重新结合电荷组分,快速度荧光衰减归属于PSⅡ反应中心组分.基于20ps模型计算的水葫芦和菠菜叶绿体PSⅡ反应中心激发能转能效率分别是87%和91%.该结果与转能效率为100%的观点不一致.实验结果支持PSⅡ反应中心电荷分裂20 ps时间常数模型.根据转能效率,水葫芦生长速度不大于菠菜生长速度,但是,水葫芦叶绿体中含有丰富的胡萝卜素成分,其单位质量叶绿体吸收光能大于单位质量菠菜叶绿体吸收的量.实验结果还暗示植物叶绿体体系传能高效,接近于100%.     

芦丁与胰蛋白酶相互作用的光谱性质研究

利用紫外光谱和荧光光谱研究了芦丁和胰蛋白酶的相互作用机制。结果表明,生理pH 7.40条件下芦丁使胰蛋白酶的紫外吸收峰增强,特征荧光峰淬灭。并利用荧光淬灭反应测得芦丁和胰蛋白酶之间结合常数KA=6.8786×104(mol/L)-1,结合位点数n=1.0173。     

ApcD, ApcF and ApcE are not required for the Orange Carotenoid Protein related phycobilisome fluorescence quenching in the cyanobacterium Synechocystis PCC 6803

In cyanobacteria, strong blue-green light induces a photoprotective mechanism involving an increase of energy thermal dissipation at the level of phycobilisome (PB), the cyanobacterial antenna. This leads to a decrease of the energy arriving to the reaction centers. The photoactive Orange Carotenoid Protein (OCP) has an essential role in this mechanism. The binding of the red photoactivated OCP to the core of the PB triggers energy and PB fluorescence quenching. The core of PBs is constituted of allophycocyanin trimers emitting at 660 or 680nm. ApcD, ApcF and ApcE are the responsible of the 680nm emission. In this work, the role of these terminal emitters in the photoprotective mechanism was studied. Single and double Synechocystis PCC 6803 mutants, in which the apcD or/and apcF genes were absent, were constructed. The Cys190 of ApcE which binds the phycocyanobilin was replaced by a Ser. The mutated ApcE attached an unusual chromophore emitting at 710nm. The activated OCP was able to induce the photoprotective mechanism in all the mutants. Moreover, in vitro reconstitution experiments showed similar amplitude and rates of fluorescence quenching. Our results demonstrated that ApcD, ApcF and ApcE are not required for the OCP-related fluorescence quenching and they strongly suggested that the site of quenching is one of the APC trimers emitting at 660nm. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

The role of ApcD and ApcF in energy transfer from phycobilisomes to PS I and PS II in a cyanobacterium

The role of the phycobilisome core components, ApcD and ApcF, in transferring energy from the phycobilisome to PS I and PS II in the cyanobacterium Synechocystis sp. PCC6803 has been investigated. The genes encoding these proteins have been disrupted in the genomes of wild type Synechocystis sp. PCC6803 and a PS II deficient mutant, PsbD1CD2^-, by inserting antibiotic resistance genes into their coding regions. Data from fluorescence emission spectra and pigment content analysis for these inactivation mutants is presented. These data suggest that both ApcD and ApcF are involved in the energy transfer route to PS II and PS I. In both cases, the energy transfer may to the reaction centres may be via the chromophore of ApcE (the L cm) or anchor polypeptide). The major route of energy transfer to both kinds of reaction centre appears to involve ApcF rather than ApcD. When both ApcF and ApcD are absent, the phycobilisomes are unable to transfer energy to either reaction centre. We suggest a model for the pathways of energy transfer from the phycobilisomes to PS I and PS II.     

Bright near-infrared fluorescence bio-labeling with a biliprotein triad

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700–770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650–700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.     

Geographic color variation of Phelotrupes auratus (Coleoptera,Geotrupidae) in the Kinki region,central Japan: A quantitative spectrophotometric analysis

We investigated geographic color variation of the beetle Phelotrupes auratus in the Kinki region of central Japan using a spectrophotometer. The reflectance spectrum of the dorsal surface of the elytron was measured for beetles collected from 23 sites. We focused on λmax(α), the wavelength at the peak (α peak) between 400 and 700 nm (human visual sensitivity range), for analyses of color variation. In populations distributed on the Kii Peninsula, in the southern part of the Kinki region, average λmax(α) values were lower (480–497 nm) than in populations distributed in the area west of Lake Biwa, the western part of the Kinki region (618–633 nm). For populations distributed in the areas south and southeast of Lake Biwa, a geographic cline of λmax(α) was observed approximately along an east–west transect, with average λmax values varying continuously from 624 to 557 nm. The easternmost populations along this cline had almost the same λmax(α) values as the populations distributed west of Lake Biwa. The coefficient of variation for λmax(α) tended to be larger in populations with intermediate averages than those with lower or higher average values.     

Structural organization of an intact phycobilisome and its association with photosystem II

Phycobilisomes (PBSs) are light-harvesting antennae that transfer energy to photosynthetic reaction centers in cyanobacteria and red algae. PBSs are supermolecular complexes composed of phycobiliproteins (PBPs) that bear chromophores for energy absorption and linker proteins. Although the structures of some individual components have been determined using crystallography, the three-dimensional structure of an entire PBS complex, which is critical for understanding the energy transfer mechanism, remains unknown. Here, we report the structures of an intact PBS and a PBS in complex with photosystem II (PSII) from Anabaena sp. strain PCC 7120 using single-particle electron microscopy in combination with biochemical and molecular analyses. In the PBS structure, all PBP trimers and the conserved linker protein domains were unambiguously located, and the global distribution of all chromophores was determined. We provide evidence that ApcE and ApcF are critical for the formation of a protrusion at the bottom of PBS, which plays an important role in mediating PBS interaction with PSII. Our results provide insights into the molecular architecture of an intact PBS at different assembly levels and provide the basis for understanding how the light energy absorbed by PBS is transferred to PSII.     

Effect of altered sterol composition on the salt tolerance of Zygosaccharomyces rouxii

To obtain mutants containing altered sterol composition and sterol contents, nystatin-resistant mutants were isolated in Zygosaccharomyces rouxii. Two of nine mutants isolated were resistant toward 20 μg of nystatin per ml, while the other seven showed resistance toward 50 μg per ml. However, the seven mutants could not grow at 35°C. TN5, a mutant of the first group, showed the same sterol composition as the wild type strain, with ergosterol and zymosterol as major sterols, whereas it contained free sterols about 70% of those of the wild type. TN1 and TN3, representative mutants of the second group, had altered sterol compositions, containing three major sterols, zymosterol, ergosta-5,7,24-trienol, and an unidentified sterol. TN1 and TN3 could not grow in YPD medium containing more than 8% NaCl, whereas TN5 grew in the same medium containing 15% NaCl after a longer lag phase than the wild type strain. TN1 and TN3, in particular TN3, when incubated in YPD medium containing 15% NaCl, leaked significant amounts of glycerol. Protoplasts of these mutants were more labile than those of the wild-type cells. These facts suggest that the amount and kind of ergosterol in the cell membrane might be concerned with the salt tolerance of Z. rouxii.     

Simultaneous cultivation of Spirulina platensis and the toxigenic cyanobacteria Microcystis aeruginosa

Mangueira Lagoon, located in the extreme south of Brazil, has water with physicochemical characteristics such as alkaline pH and carbonate levels propitious for the growth of the cyanobacterium Spirulina platensis. Previously published studies have shown that Mangueira Lagoon water supplemented with small quantities of carbon and nitrogen is suitable for S. platensis cultivation and can significantly reduce production costs. We studied mixed cultures of Spirulina platensis and the toxic cyanobacterium Microcystis aeruginosa using a 2(3) factorial design in which the three factors were the initial biomass concentration of S. platensis and M. aeruginosa and the type of culture medium (100% Zarrouk's medium or 80% Mangueira Lagoon water plus 20% Zarrouk's medium). The highest S. platensis maximum specific growth rate (mu(max)) occurred in the culture with the highest M. aeruginosa biomass concentration and when undiluted culture medium was used (micro(max) = 0.283 d(-1)). The highest M. aeruginosa specific death rate (k) was obtained in the presence of S. platensis (k = 0.555 d(-1)) and was independent of the initial M. aeruginosa biomass concentration and culture medium, demonstrating that S. platensis cultures are not susceptible to contamination by M. aeruginosa. The culture medium had no significant influence (p > 0.05) on S. platensis micro(max) values, indicating that production costs could be reduced by using a medium consisting of 80% Mangueira Lagoon water plus 20% Zarrouk's medium.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Reconstitution of phycobilisome core-membrane linker, LCM, by autocatalytic chromophore binding to ApcE

The core-membrane linker, LCM, connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane. Genes coding for the apoprotein, ApcE, from Nostoc sp. PCC 7120 and for a C-terminally truncated fragment ApcE(1-240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli. Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins. If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker LCM, demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE. The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling. The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-alpha84 lyases, indicates that ApcE constitutes a third type of bilin:biliprotein lyase.