Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M_r 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP_CM since the large linker polypeptide L_CM was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP_C, M_r≥1000000) consisting of both AP_CM and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP_CM can be isolated from phycobilisomes of Anabaena variabilis. This is evidence that AP_CM is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

Protective dissipation of excess absorbed energy by photosynthetic apparatus of cyanobacteria: role of antenna terminal emitters

Two mechanisms of photoprotective dissipation of the excessively absorbed energy by photosynthetic apparatus of cyanobacteria are described that divert energy from reaction centers. Energy dissipation, monitored as nonphotochemical fluorescence quenching, occurs at different steps of energy transfer within the phycobilisomes or core antenna of photosystem I. Although these mechanisms differ significantly, in both cases, energy dissipates mainly from terminal emitters: allophycocyanin B or core membrane linker protein (L_CM) in phycobilisomes, or the longest-wavelength chlorophylls in photosystem I antenna. It is supposed that carotenoid-induced energy dissipation in phycobilisomes is triggered by light-induced transformation of the nonquenched state of antenna into quenched state due to conformation changes caused by orange carotinoid-binding protein (OCP)–phycobilisome interaction. Fluorescence of the longest-wavelength chlorophylls of photosystem I antenna is strongly quenched by P700 cation radical or by P700 triplet state, dependent on redox state of the acceptor side cofactors of photosystem I.     

Core substructure in Mastigocladus laminosus phycobilisomes

Three allophycocyanin complexes were separated by gel electrophoresis, isoelectric focusing and ion exchange chromatography from a low molecular fraction (M_r 100–150000) of partially dissociated phycobilisomes of Mastigocladus laminosus: A. (^APAP); B. (^*AP₂ ^AP₂ ^AP*AP) · L _C ¹⁰ ; and C. (^*APAPBAP₂ ^AP*AP) · L _C ¹⁰ . According to their fluorescence emission maximum at room temperature the complexes A., B. and C. are designated AP 660, AP 664 and AP 680. The different subunits of the AP complexes have apparent molecular weights of M_r 18500 ^*AP, 18200 ^APB, 18000 ^AP, 17000 ^AP and 16500 ^*AP. This hitherto unrecognized microheterogeneity within the AP subunits of complexes B. and C. of Mastigocladus laminosus phycobilisomes could also be demonstrated and confirmed with the two phycocyanin complexes PC 642 and PC 646. PC 642 is characterized by a L _R ¹¹ linker polypeptide.Abbreviations AP allophycocyanin - PC phycocyanin - PEC phycoerythrocyanin - PE phycoerythrin - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - pI isoelectric point - M_r apparent molecular weight - TMED tetramethylethylenediamine - APS ammonium persulphate - SDS sodium dodecylsulphate - O.D. optical density A preliminary account of this work has been presented at the Embo Workshop on Oxygenic and Anoxygenic Electron Transport Systems in Cyanobacteria (Blue-green Algae) in Cape Sounion, Greece, 20–25 September 1987     

Adaptation of the Photosynthetic Apparatus of Anacystis nidulans to Irradiance and CO2-Concentration

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m², and supplied with 0.03 and 3 % CO₂ enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO₂, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO₂ concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L³⁰_R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO₂ adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO₂ adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L⁶⁰_CM and L⁷⁵_CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.     

Phycobilisome from Anabaena Variabilis Kütz. and its Model Conjugates

The model conjugates phycocyanin-allophycocyanin (C-PC-APC) and phycoerythrocyanin-phycocyanin-allophycocyanin (PEC-C-PC-APC) were synthesized by using a heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The rod-core complex (αβ)₆ ^PCL_RC ²⁷(αβ)₃ ^APCL_C ^8.9 and phycobilisomes were separated from Anabaena variabilis. Energy transfer features for the conjugates and the complexes were compared. The absorption and fluorescence emission spectra indicated that the linker-peptides mediate interaction of phycobiliproteins and prompt energy transfer. The energy transfer in the conjugates was detected by fluorescence emission spectra and confirmed by the addition of dithiothreitol. The conjugates may be used as models for studying the energy transfer mechanism in phycobilisomes. This revised version was published online in September 2006 with corrections to the Cover Date.     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.     

UV-induced phycobilisome dismantling in the marine picocyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. WH8102

The marine picocyanobacterium Synechococcus sp. WH8102 was submitted to ultraviolet (UV-A and B) radiations and the effects of this stress on reaction center II and phycobilisome integrity were studied using a combination of biochemical, biophysical and molecular biology techniques. Under the UV conditions that were applied (4.3 W m⁻² UV-A and 0.86 W m⁻² UV-B), no significant cell mortality and little chlorophyll degradation occurred during the 5 h time course experiment. However, pulse amplitude modulated (PAM) fluorimetry analyses revealed a rapid photoinactivation of reaction centers II. Indeed, a dramatic decrease of the D1 protein amount was observed, despite a large and rapid increase in the expression level of the psbA gene pool. Our results suggest that D1 protein degradation was accompanied (or followed) by the disruption of the N-terminal domain of the anchor linker polypeptide L_CM, which in turn led to the disconnection of the phycobilisome complex from the thylakoid membrane. Furthermore, time course analyses of in vivo fluorescence emission spectra suggested a partial dismantling of phycobilisome rods. This was confirmed by characterization of isolated antenna complexes by SDS-PAGE and immunoblotting analyses which allowed us to locate the disruption site of the rods near the phycoerythrin I—phycoerythrin II junction. In addition, genes encoding phycobilisome components, including α-subunits of all phycobiliproteins and phycoerythrin linker polypeptides were all down regulated in response to UV stress. Phycobilisome alteration could be the consequence of direct UV-induced photodamages and/or the result of a protease-mediated process.     

Distinct roles of CpcG1-phycobilisome and CpcG2-phycobilisome in state transitions in a cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

State transitions in cyanobacteria regulate the relative energy transfer from phycobilisome to photosystem I and II. Although it has been shown that phycobilisome mobility is essential for phycobilisome-dependent state transitions, the biochemical mechanism is not known. Previously we reported that two distinct forms of phycobilisome are assembled with different CpcG copies, which have been referred to as “rod-core linker,” in a cyanobacterium Synechocystis sp. PCC 6803. CpcG2-phycobilisome is devoid of a typical central core, while CpcG1-phycobilisome is equivalent to the conventional phycobilisome supercomplex. Here, we demonstrated that the cpcG1 disruptant has a severe specific defect in the phycobilisome-dependent state transition. However, fluorescence recovery after photobleaching measurements showed no obvious difference in phycobilisome mobility between the wild type and the cpcG1 disruptant. This suggests that both CpcG1 and CpcG2 phycobilisomes have an unstable interaction with the reaction centres. However, only CpcG1 phycobilisomes are involved in state transitions. This suggests that state transitions require the phycobilisome core.     

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.     

The phycobilisome core-membrane linkers from Synechocystis sp. PCC 6803 and red-algae assemble in the same topology

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (L_CM) specifically organizes the allophycocyanin subunits in the PBS’s core. But, the topology of L_CM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of L_CM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes L_CM, that the cyanobacterial and red-algal L_CM topologies are actually the same. We found that removing the top cylinder linker domain in L_CM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal L_CM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.     

Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR_L) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR_L accumulated. FNR_L has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR_L and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.     

Noncovalent Intermolecular Forces in Phycobilisomes of Porphyridium cruentum

Using sensitized fluorescence as a measure of intactness of phycobilisomes isolated from Porphyridium cruentum, the effects of various environmental perturbations on phycobilisome integrity were investigated. The rate of phycobilisome dissociation in 0.75 ionic strength sodium salts proceeds in the order: SCN⁻ > NO₃⁻ > Cl⁻ > C₆H₅O₇³⁻ > SO₄²⁻ > PO₄³⁻, as predicted from the lyotropic series of anions and their effects on hydrophobic interactions in proteins. Similarly, increasing temperature (to 30 C) and pH values approaching the isoelectric points of the biliproteins stabilize phycobilisomes. Deuterium substitution at exchangeable sites on the phycobiliproteins decreases the rate of phycobilisome dissociation, while substitution at nonexchangeable sites increases rates of dissociation. It is concluded that hydrophobic intermolecular interactions are the most important forces in maintaining the phycobilisome structure. Dispersion forces also seem to contribute to phycobilisome stabilization. The adverse effects of electrostatic repulsion must not be ignored; however, it seems that the requirement of phycobilisomes of high salt concentrations is not simply countershielding of charges on the proteins.     

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems’ activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions—P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L_CM is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L_CM may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.     

Large reallocations of carbon,nitrogen, and photosynthetic reductant among phycobilisomes,photosystems, and Rubisco during light acclimation in <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> strain PCC7942 are constrained in cells under low environmental inorganic carbon

Synechococcus elongatus strain PCC7942 cells were grown in high or low environmental concentrations of inorganic C (high-C_i, low-C_i) and subjected to a light shift from 50 µmol m^–2 s^–1 to 500 µmol m^–2 s^–1. We quantified photosynthetic reductant (O₂ evolution) and molar cellular contents of phycobilisomes, PSII, PSI, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through the light shift. Upon the increase in light, small initial relative decreases in phycobilisomes per cell resulted from near cessation of phycobilisome synthesis and their dilution into daughter cells. Thus, allocation of reductant to phycobilisome synthesis dropped fivefold from pre- to post-light shift. The decrease in phycobilisome synthesis liberated enough material and reductant to allow a doubling of Rubisco and up to a sixfold increase in PSII complexes per cell. Low-C_i cells had smaller initial phycobilisome pools and upon increased light; their reallocation of reductant from phycobilisome synthesis may have limited the rate and extent of light acclimation, compared to high-C_i cells. Acclimation to increased light involved large reallocations of C, N, and reductant among different components of the photosynthetic apparatus, but total allocation to the apparatus was fairly stable at ca. 50% of cellular N, and drew 25–50% of reductant from photosynthesis.     

Localization and quantitation of chloroplast enzymes and light-harvesting components using immunocytochemical methods

Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD L_cm linker) and for ATP synthase (β subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments.     

What can we learn about the lipid vesicle structure from the small-angle neutron scattering experiment?

Small-angle neutron scattering (SANS) on the unilamellar vesicle (ULV) populations (diameter 500 and 1,000 Å) in D₂O was used to characterize lipid vesicles from dimyristoylphosphatidylcholine (DMPC) at three phases: gel L_β′, ripple P_β′ and liquid L_α. Parameters of vesicle populations and internal structure of the DMPC bilayer were characterized on the basis of the separated form factor (SFF) model. Vesicle shape changes from nearly spherical in the L_α phase to elliptical in the P_β′ and L_β′ phases. This is true for vesicles prepared via extrusion through pores with the diameter 500 Å. Parameters of the internal bilayer structure (thickness of the membrane and the hydrophobic core, hydration and the surface area of the lipid molecule) were determined on the basis of the hydrophobic–hydrophilic (HH) approximation of neutron scattering length density across the bilayer ρ(x) and of the step function (SF) approximation of ρ(x). DMPC membrane thickness in the L_α phase (T=30°C) demonstrates a dependence on the membrane curvature for extruded vesicles. Prepared via extrusion through 500 Å diameter pores, vesicle population in the L_α phase has the following characteristics: average value of minor semi-axis 266±2 Å, ellipse eccentricity 1.11±0.02, polydispersity 26%, thickness of the membrane 48.9±0.2 Å and of the hydrophobic core 19.9±0.4 Å, surface area 60.7±0.5 Ų and number of water molecules 12.8±0.3 per DMPC molecule. Vesicles prepared via extrusion through pores with the diameter 1,000 Å have polydispersity of 48% and membrane thickness of 45.5±0.6 Å in the L_α phase. SF approximation was used to describe the DMPC membrane structure in L_β′ (T=10°C) and P_β′ (T=20°C) phases. Extruded DMPC vesicles in D₂O have membrane thickness of 49.6±0.5 Å in the L_β′ phase and 48.3±0.6 Å in the P_β′ phase. The dependence of the DMPC membrane thickness on temperature was restored from the SANS experiment.     

Learned changes in the complexity of movement organization during multijoint, standing pulls

 This paper tests the hypothesis that the central nervous system (CNS) learns to organize multijoint movements during a multijoint ‘bouncing pull’ task such that, after practice, motion of the anterior-posterior center of mass (CM_AP) more closely resembles that of a conservative, one degree of freedom (DF), inverted pendulum model. The task requires standing human subjects to produce precise peak pulling forces on a handle while maintaining balance – goals that can be easily accomplished if movement is organized as in the model. Ten freely standing subjects practiced making brief, bouncing pulls in the horizontal direction to target forces (20–80% of maximum) for 5 days. Pulling force, body kinematic and force plate data were recorded. An eight-segment analysis determined sagittal-plane CM motion. We compared the effects of practice on the regression-based fit between actual and model-simulated CM_AP trajectories, and on measures of CM_AP phase plane symmetry and parameter constancy that the model predicts. If the CNS learns to organize movements like the inverted pendulum model, then model fit should improve and all other measures should approach zero after practice. The fit between modeled and actual CM_AP motion did not improve significantly with practice, except for moderate force pulls. Nor did practice increase phase plane symmetry or parameter constancy. Specifically, practice did not decrease the differences between the pre-impact and rebound positions or speeds of the CM_AP, although speed difference increased with pulling force. CM_AP at the end of the movement was anterior to its initial position; the anterior shift increased after practice. Differences between the pre-pull and balance-recovery ankle torque (T _A) impulses were greater on day 5 and correlated with the anterior shift in CM_AP. These results suggest that practice separately influenced the force production and balance recovery phases. A modified model with damping could not explain the observed behaviors. A modified model using the actual time-varying T_A profiles improved fit at lower force levels, but did not explain the increased postural shift after practice. We conclude that the CNS does not learn to organize movements like the conservative, inverted pendulum model, but rather learned a more complex form of organization that capitalized on more time-varying controls and more intersegmental dynamics. We hypothesize that at least one additional DF and at least one time-varying parameter will be needed to explain fully how the CNS learns to organize multijoint, bouncing pulls made while standing. Received: 9 January 1997 / Accepted in revised form: 27 May 1997     

Biliprotein assembly in the hemidiscoidal phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus Cohn. Characterization of dissociation products with special reference to the peripheral phycoerythrocyanin-phycocyanin complexes

The dissociation products of isolated phycobilisomes of Mastigocladus laminosus were separated and analyzed by ultracentrifugation and, in part, by isoelectric focusing. With the exception of the allophycocyanin core, the sedimentation constants of peripheral phycocyanin- and phycoerythrocyanin-phycocyanin complexes lay in the range of 6 to 17S. The latter was represented by a 17S aggregate of two hexameric phycocyanins (dodecamer, dipartite unit). A complex with an absorption maximum at 610 nm (phycocyanin) and a shoulder at 580 nm (phycoerythrocyanin), a fluorescence emission maximum at 645 nm and a sedimentation constant of 11 S is described as a heterogeneously composed hexamer of ()₃-phycoerythrocyanin-()₃-phycocyanin. It was stable under extended dissociation in the cold and under isoelectric focusing. An aggregate of 14 S with an absorption maximum at 576 nm and a shoulder in the fluorescence emission spectrum at 625 nm (phycoerythrocyanin) in addition to the maximum at 645 nm (phycocyanin) is interpreted as a polar phycoerythrocyanin/ phycoerythrocyanin-phycocyanin complex. Combining these complexes with phycocyanin dodecamers creates peripheral rods of the phycobilisome. A proposal of the phycobiliprotein distribution within the phycobilisome of M. laminosus is presented.Abbreviations APC allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin     

Physiological significance of P2X receptor-mediated vasoconstriction in five different types of arteries in rats

P2X₁ receptors, the major subtype of P2X receptors in the vascular smooth muscle, are essential for α,β-methylene adenosine 5′-triphosphate (α,β-MeATP)-induced vasoconstriction. However, relative physiological significance of P2X₁ receptor-regulated vasoconstriction in the different types of arteries in the rat is not clear as compared with α₁-adrenoceptor-regulated vasoconstriction. In the present study, we found that vasoconstrictive responses to noncumulative administration of α,β-MeATP in the rat isolated mesenteric arteries were significantly smaller than those to single concentration administration of α,β-MeATP. Therefore, we firstly reported the characteristic of α,β-MeATP-regulated vasoconstrictions in rat tail, internal carotid, pulmonary, mesenteric arteries, and aorta using single concentration administration of α,β-MeATP. The rank order of maximal vasoconstrictions for α,β-MeATP (E _{max·α,β-MeATP}) was the same as that of maximal vasoconstrictions for noradrenaline (E _max·NA) in the internal carotid, pulmonary, mesenteric arteries, and aorta. Moreover, the value of (E _{max·α,β-MeATP}/E _max·KCl)/(E _max·NA/E _max·KCl) was 0.4 in each of the four arteries, but it was 0.8 in the tail artery. In conclusion, P2X₁ receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries, and aorta, but much greater in the tail artery, suggesting its special role in physiological function.     

Structure and mutation of a gene encoding a Mr 33000 phycocyanin-associated linker polypeptide

The gene encoding a phycocyanin-associated linker polypeptide of M_r 33000 from the cyanobacterium Synechococcus sp. PCC 7002 was found to be located adjacent and 3 to the genes encoding the and subunits of phycocyanin. The identity of this gene, designated cpcC, was proven by matching the amino-terminal sequence of the authentic polypeptide with that predicted by the nucleotide sequence. A cpcC mutant strain of this cyanobacterium was constructed. The effect of the mutation was to prevent assembly of half the total phycocyanin into phycobilisomes. By electron microscopy, phycobilisomes from this mutant were shown to contain rod substructures composed of a single disc of hexameric phycocyanin, as opposed to two discs in the wild type. It was concluded that the M_r 33000 linker polypeptide is required for attachment of the core-distal phycocyanin hexamer to the core-proximal one. Using absorption spectra of the wild type, CpcC^–, and phycocyanin-less phycobilisomes, the in situ absorbances expected for specific phycocyanin-linker complexes were calculated. These data confirm earlier findings on isolated complexes regarding the influence of linkers on the spectroscopic properties of phycocyanin.Abbreviations PC phycocyanin - PEC phycoerythrocyanin - AP allophycocyanin - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Linker polypeptides are abbreviated according to Glazer (1985). L _infX ^supY refers to a linker having a mass Y, located at a position X in the phycobilisome, where X can be R (rod), RC (rod or core), C (core) or CM (core to membrane). When necessary, the abbreviation for a linker is appended with that of its associated phycobiliprotein. Thus, L _infR ^sup34.5PEC is a rod linker of M_r 34 500 that is associated with phycoerythrocyanin