条斑紫菜藻红、藻蓝蛋白逐级放大的纯化工艺

采用“破碎-盐析-层析”的方法纯化条斑紫菜藻胆蛋白,并在提取规模上逐步放大。首先在综合比较凝胶层析去盐效率后,从Sephadex G-25、G-100、S-300和CL-6B中选择G-25作为实验流程中的去盐填料,其次将提取流程的初试原料条斑紫菜量逐步放大,选取了1g、20g和400g三个量,结果表明随着初试紫菜量逐步放大,最终所得藻胆蛋白中吸收光谱纯度>3.2的蛋白产率依次提高,其中400g冻干紫菜的藻红蛋白产率为0.323％,藻蓝蛋白产率为0.148％。由此认为该实验工艺流程具有规模放大的潜力,这为高纯度藻胆蛋白的规模生产提供了一条可行的方案。     

Native and in vitro-associated phycobilisomes of Nostoc sp. Composition,energy transfer,and effect of antibodies

The relationship of the structure and function of the light-harvesting antennae in the blue-green alga Nostoc sp. was further elucidated by reconstitution experiments. Separated phycoerythrin-phycocyanin complexes and allophycocyanin fractions were reassociated as described earlier (Canaani, O., Lipschultz, C.A. and Gantt, E. (1980) FEBS Lett. 115, 225–229) into functional phycobilisomes with a 70% yield. Native and reassociated physobilisomes had molar ratios of about 1.4:1.1:1.0 of phycoerythrin:phycocyanin:allophycocyanim. Energy transfer was demonstrated by their fluorescence emission maximum at approx. 675 nm (20°C), and their excitation spectra (emission wavelength 680 nm) which reflected the contribution of the three constitutive phycobiliproteins. Scans of Coomassie blue-stained SDS-polyacrylamide gels showed that the polypeptide composition of native and reassociated phycobilisomes was virtually indistinguishable. Reassociation of phycobilisomes was dependent on the interaction of allophycocyanin and phycocyanin, because it could be blocked with antisera to phycocyanin and allophycocyanin, but not to phycoerythrin. In addition, reassociation did not occur when a 31 000 Da polypeptide, which is part of the phycoerythrin-phycocyanin complex, was reduced in size (by 4000 Da). These results suggest that at least two domains are required for functional reassociation of phycobilisomes involving phycocyanin and allophycocyanin.     

Energy Transfer from Phycobilisomes to Photosystems of Nostoc flagelliforme Born. et Flah. During the Rewetting Course and Its Physiological Significance

During the non-frost season, the condensation of dew makes Nostocflagelliforme Born. et Flah., a highly drought-tolerant terrestrial cyanobacterium, frequently undergo rehydration-dehydration.Rehydration begins in the dark at night. After rewetting in the dark, photochemical activity and the structure of photosystem (PS) Ⅱ were not recovered at all; the structure of PSⅠ, energy transfer in phycobilisomes, and energy transfer from phycobilisomes to PSⅠ were recovered within 5 min, as in the light. The recovery of energy transfer from phycobilisomes to PSⅡ was light dependent and energy transfer from phycobilisomes to PSⅡ was only partially recovered in the dark. These results suggest that the two-trigger control (water and light) of photosynthetic recovery may make N. flagelliforme avoid unnecessary energy consumption and, at the same time, the partial recovery of energy transfer from phycobilisomes to PSⅡ in the dark could help N. flagelliforme accumulate more photosynthetic products during the transient period of rehydrationdehydration.     

Effects of proteinase K on the energy transfer between phycobiliproteins in phycobilisomes

Spectral changes in fluorescence of phycobilisomes (PBS) of A. variabilis treated with proteinase K were studied at room and liquid nitrogen temperature. In control PBS, the relative yield of 77 K fluorescence of F686 was very high, and those of F655 and F666 were low. In PBS treated with proteinase K for less than 1 h, F686 decreased, and F655 and F666 increased. In PBS treated with proteinase K for 2 h, F655 was the main peak of fluorescence emission, F686 was the second peak, the fluorescence emission peak of F666 disappeared. In PBS treated with proteinase K for more than 8 h, F655 showed only one fluorescence emission peak.We suggested that phycobiliporteins in the PBS of A. variabilis constitute an energy transfer chain, shown as follows:{fx91-1}The linkages between APC and APC-B, C-PC and APC, and C-PC and APC-B had different sensitivity towards proteinase K.     

Core substructure in cyanobacterial phycobilisomes

The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (alpha AP beta AP)3; B, (alpha AP beta AP)3 .10K; C, (alpha APB1 alpha AP2 beta AP3).10K; D, (alpha AP beta AP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.     

Abundance and pigment type composition of picocyanobacteria in Barguzin Bay, Lake Baikal

In Lake Baikal, picocyanobacteria are the most important primary producers during the summer. Freshwater picocyanobacteria are discriminated into either the phycoerythrin (PE)-rich or the phycocyanin (PC)-rich types according to their pigment composition. The distributions of these two types of picocyanobacteria were investigated in Barguzin Bay. The PC-rich type accounted for >98% of the total picocyanobacteria at the station near the shore of the bay where river water flows directly in. In the offshore area of the lake, all of the picocyanobacteria cells were of the PE-rich type. In addition, the occurrence of the PC-rich type was restricted to the station, where the attenuation coefficient exceeded 0.25 m⁻¹. Near the shore, where the turbidity was high (>1 NTU), the cell densities of both the PE- and PC-rich types increased away from the river mouth. This indicates that the PC-rich type cells grow near the shore of the bay where turbidity is high. Since the PC-rich type could not grow well when cells were incubated in offshore lake water, restricted distribution of the PC-rich type could also be explained by their growth capability. The present study clearly demonstrated the shift in the pigment type composition of picocyanobacteria from the coastal to the pelagic zone of Lake Baikal. The co-existence of the two pigment types probably enables the abundance of the picocyanobacterial community to be stable over a broader range of environmental conditions than would be possible for a single pigment type.     

Energy transfer kinetics of phycoerythrocyanin trimer from cyanobacterium Anabaena variabilis (II)

The excitation energy transfer processes in trimeric PEC have been studied by using steady-state and time-resolved fluorescence spectra techniques in detail. The results indicate that the energy transfer processes should take place between α₈₄-PVB and β₈₄-or β₁₅₅-PCB chromophores with the time constants 34.7 ps and 175–200 ps individually; in contrast with monomeric PEC, from time-resolved fluorescence anisotropic spectrum technique, the decay constant of 45 ps which was assigned to the energy transfer time among three β₈₄-PCB chromophores was observed and the energy levels of β₈₄-and/or β₁₅₅-PCB chromophores were confirmed to turn over in trimeric PEC. Project supported by the National Natural Science Foundation of China (Grant No. 39670065).     

Energy transfer kinetics of phycoerythrocyanin trimer from cyanobacterium Anabaena variabilis (II)

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Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome

Anabaena sp. PCC 7120 mutants defective in phycobiliprotein biosynthesis or phycobilisome assembly were generated by transposon mutagenesis. Four mutants with grossly reduced content of the major phycobiliprotein, phycocyanin, were found to have insertions within the cpcBACDEFG1G2G3G4 operon coding for phycocyanin biosynthesis and assembly. The insertion in mutant B646 separated the promoter from the open reading frames and eliminated production of the phycocyanin (CpcA) and (CpcB) subunits. Insertion in cpcC in mutant B642 eliminated production of the L³⁶ _Rlinker polypeptide required for assembly of phycocyanin into the distal discs of the phycobilisome rod substructures. Mutants B64328 and B64407 had insertions, respectively, in cpcE and cpcF, genes coding for the subunits of the heterodimeric lyase which catalyzes the attachment of phycocyanobilin to the phycocyanin apo- subunit. Mutant SB12, often unable to survive under low light, was found to have an insertion in the apcE gene coding for the large core-membrane linker (L¹²⁸ _CM) that provides the scaffold for assembly of the phycobilisome core. DNA sequencing 3 of apcE revealed genes apcABC, coding for the and subunits of allophycocyanin and for the small core linker L^7.8 _C. Amino acid sequence comparisons showed that the ApcA and ApcB proteins are 37% identical and that each of these polypeptides is highly similar to corresponding polypeptides from the distantly related filamentous strains Calothrix sp. PCC7601 and Mastigocladus laminosus.     

Visualization of excitation energy transfer processes in plants and algae

Development of the time-resolved fluorescence spectroscopy in the pico-second time range and its application to the energy transfer processes in many photosynthetic organisms is reviewed here. This method enabled visualization of energy transfer processes by three-dimensional expression of fluorescence spectra and discrimination of kinetic components and spectral components. The second generation of the ultrafast fluorescence spectroscopy is the femto-second (fs) fluorescence up-conversion, and this has enabled analyses of the transfer processes from carotenoids to chlorophylls with a resolution of less than 100 fs. For future progress, a further development of the spectroscopy is indispensable as well as structural data at atomic resolution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fluorescence from low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at physiological temperatures

Fluorescence spectra from Photosystem I (PS I) are measured from 25 to –5 °C on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Emission from antenna chlorophylls (Chls) with energy levels below that of the reaction center, or low-energy Chls (LE Chls), is resolved verifying their presence at physiological temperatures. The 25°C spectrum is characterized by peaks at 688 and 715 nm. As temperature decreases, fluorescence at 688 nm decreases while at 715 nm it increases. The total fluorescence yield does not change. The temperature dependent spectra are fit to a sum of two basis spectra. At 25°C, the first basis spectrum has a major peak at 686 nm and a minor peak at 740 nm. This is attributed to fluorescence from the majority or bulk antenna Chls. The second basis spectrum has a major peak at 712 nm, with shoulders at 722 and 770 nm. It characterizes fluorescence from a small number of LE Chls. A progressive shift to the red in the fluorescence spectra occurs as the temperature is decreased. The temperature dependence in the relative amount of fluorescence from the bulk and LE Chls is fit using a two-component energy transfer model at thermal equilibrium.     

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

Spectral resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 through direct excitation

We have measured fluorescence spectra from Photosystem I (PS I) on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 at room temperature as a function of excitation wavelength. Our data show a gradual enhancement of long-wavelength fluorescence at 710 nm as the excitation wavelength is increased from 695 to 720 nm. This verifies the presence of low-energy chlorophylls (LE Chls), antenna Chls with energy levels below that of the primary electron donor, P700. The change in fluorescence with excitation wavelength is attributed to the finite time it takes for equilibration of excitations between the bulk and LE Chls. The spectra were deconvoluted into the sum of two basis spectra, one an estimate for fluorescence from the majority or bulk Chls and the other, the LE Chls. The bulk Chl spectrum has a major peak at 688 nm and a lower amplitude vibrational band around 745 nm and is assumed independent of excitation wavelength. The LE Chl spectrum has a major peak at 710 nm, with shoulders at 725 and 760 nm. The relative amplitude of emission at the vibrational side bands increases slightly as the excitation wavelength increases. The ratio of the fluorescence yields from LE Chls to that from bulk Chls ranges from 0.3 to 1.3 for excitation wavelengths of 695 to 720 nm, respectively. These values are consistent with a model where the LE Chls are structurally close to P700 allowing for direct transfer of excitations from both the bulk and LE Chls to P700.     

Genetic diversity among Synechococcus spp. (cyanobacteria) isolated from the pelagial of Lake Constance

Abstract: Seven phycoerythrin (PE)-rich and six phycocyanin (PC)-rich unicellular cyanobacteria of the Synechococcus type were isolated from the pelagial of Lake Constance. The genetic diversity among the isolates was evaluated using restriction fragment length polymorphism (RFLP) within the psbA gene family. Nine out of 13 isolates exhibit different DNA structures in the probed areas and, furthermore, they differ from morphologically similar strains collected from other lakes. The data set does not support a principal distinction between PC-rich and PE-rich strains but it reveals less heterogeneity in the coding region of the psbA genes among PE-rich isolates than among PC-rich isolates. The isolation of distinct strains in different seasons suggests species diversity and seasonal occurrence of Synechococcus spp. in Lake Constance.     

Time-dependent alterations in the antenna pigment-protein complex by mercury ions in the cyanobacterium Spirulina platensis

We have shown that mercury affects energy transfer in Spirulina platensis. It inhibits energy transfer from phycocyanin to chlorophyll a by specifically bleaching the -84 chromophore of the chromo protein, phycocyanin (PC), in the cyanobacterium. This effect is observed during short-term exposure of cells to Hg²⁺ ions. Upon long-term (12 h) exposure, mercury at low concentrations (1–2.5 m) causes the gradual degradation of the polypeptide (22 kDa) of the PC of phycobilisomes in this cyanobacterium. The effect of mercury on this polypeptide is significant compared with the other phycobiliproteins.     

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24–48 h according to various steps. For O₂ evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.Abbreviations APC allophycocyanin - CAP chloroamphenicol - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CP-47 chlorophyll-binding Photosystem II protein of 47 kDa - EF exoplasmic face - PBS phycobilisome - PC phycocyanin - PS Photosystem     

假根羽藻外周天线色素分子间能量传递

假根羽藻外周天线捕光色素蛋白复合物(L ight-harvesting Comp lex II,LHC II)在不同聚集态的情况下,它所包含色素分子间的能量传递是不同的。采用荧光发射光谱和激发光谱技术对不同聚集态(单体、三聚体和寡聚体)的LHC II进行研究,发现三聚体中色素分子间的能量传递效率比较高,单体要小一些。520 nm激发下,类胡萝卜素分子向叶绿素a分子的能量传递效率:三聚体约为64%、单体约为56%;650 nm激发下,叶绿素b分子向叶绿素a分子的能量传递效率:三聚体约为89%、单体约为78%。寡聚体的能量传递要复杂些,从光谱分析出它包含两种不同吸收光谱特性的叶绿素b分子,吸收峰分别为480 nm和468 nm,其中蓝区吸收峰为480 nm的叶绿素b分子向发射685 nm荧光的叶绿素a分子的能量传递效率要小于75%。     

Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant

The molecular architectures of photosynthetic complexes are rapidly becoming available through the power of X-ray crystallography. These complexes are comprised of antenna complexes, which absorb and transfer energy into photochemical reaction centers. Most reaction centers, found in both oxygenic and non-oxygenic species, are connected to transmembrane chlorophyll containing antennas, and the crystal structures of these antennas contain information on the structure of the entire complex as well as clear indications on their modes of functional association. In cyanobacteria and red alga, most of the Photosystem II associated light harvesting is performed by an enormous (3–7 MDa) membrane attached complex called the phycobilisome (PBS). While the crystal structures of many isolated components of different PBSs have been determined, the structure of the entire complex as well as its manner of association with Photosystem II can only be suggested. In this review, the structural information obtained on the isolated components will be described. The structural information obtained from the components provides the basis for the modeled reconstruction of this giant complex.