Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta)

The genes encoding the and subunits of allophycocyanin, phycocyanin and phycoerythrin from the red alga Aglaothamnion neglectum were isolated and characterized. While the operons containing the different phycobiliprotein genes are dispersed on the plastid genome, the genes encoding the and subunits for each phycobiliprotein are contiguous. The subunit gene is 5 for both the phycocyanin and phycoerythrin operons, while the subunit gene is 5 for the allophycocyanin operon. The amino acid sequences of A. neglectum phycobiliproteins, as deduced from the nucleotide sequences of the genes, are 65–85% identical to analogous proteins from other red algae and cyanobacteria. The conserved nature of the plastid-encoded red algal and cyanobacterial phycobiliprotein genes supports the proposed origin of red algal plastids from cyanobacterial endosymbionts.Many environmental factors effect phycobilisome biosynthesis. The effect of both nutrient availability and light quantity on the level of A. neglectum phycobiliprotein subunits and the mRNA species encoding those subunits is described.     

Light-harvesting phycobiliprotein pigments of the red algaLeptosomia simplex from the Antarctic

Summary By means of Sephadex G-100 chromatography method, the phycobiliprotein content of Antarctic red alga-Leptosomia simplex was studied. The phycobiliproteins, R-phycoerythrin, R-phycocyanin and allophycocyanin were identified. The dominant pigment in this group was found to be R-phycoerythrin.Part VII in the series Studies on phycobiliproteins in algae     

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.     

Subunit composition of the zinc proteins alpha- and beta-lipovitellin from chicken

Chicken - and -lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per -lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per -lipovitellin, respectively. The subunits of -lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of - and -lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from - and -lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of - and -Lv, respectively, and two peptides of the 30-kDa subunit of - and -lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of - and -lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of -lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Notes on the Mechanism of ATP Synthesis

The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and P_i assumes that all states of the F₁ portion of the ATP synthase have subunits in tight, loose, and open conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional subunit conformation, closed, observed in the rat liver structure overcomes these difficulties.     

Structure and molecular organization of the photosynthetic accessory pigments of cyanobacteria and red algae

Summary Cyanobacteria (blue-green algae) and Rhodophyta (red algae) contain high concentrations of photosynthetic accessory pigments (phycobiliproteins) which trap light energy in the region between 400 and 650 nm. The electronic excitation energy is then transferred along a chain of these pigments to the reaction center chlorophyll of Photosystem II by a radiationless induced resonance process.Unlike the protein-chlorophyll complexes in the photosynthetic lamellae, the phycobiliproteins are readily soluble in aqueous solution, can be isolated in a variety of assembly forms, and crystallize readily. These properties facilitate the study of the structure of these proteins by chemical, physical, and immunological methods, as well as by X-ray diffraction and electron microscopy.The brilliantly colored phycobiliproteins are a homologous family of conjugated proteins of differing spectroscopic properties. The basic structural unit in these proteins is a monomer of 30,000–40,000 daltons made up of two dissimilar polypeptide chains, and . Each subunit carries covalently linked tetrapyrrole prosthetic groups related to the bile pigment biliverdin.The distinctive spectroscopic properties of each phycobiliprotein are a consequence of the chemical structure of the bile pigment it carries, and of the influence of the conformation and aggregation state of the protein on the spectra of these prosthetic groups. In vivo, the phycobiliproteins are organized into particles, phycobilisomes, attached in a regular array to the outer surface of the photosynthetic lamellae. Studies on phycobilisomes, and on intact cells, indicate the following pathway of energy transfer.Phycoerythrin Phycocyanin (_max 560 nm) (_max 620 nm) Allophycocyanin Allophycocyanin B (_max 650 nm)(_max 671 nm) Chlorophyll a (_max 680 nm)The amounts of the various phycobiliproteins in the cell are influenced by the intensity and energy distribution of the incident radiation. The phenomena of intensity adaptation and complementary chromatic adaptation yield insights into the structure of phycobilisomes and the molecular basis of the plasticity of the structure of this light-harvesting system.Invited article.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

AKIN<Emphasis Type="Italic">β</Emphasis>3, a plant specific SnRK1 protein,is lacking domains present in yeast and mammals non-catalytic <Emphasis Type="Italic">β</Emphasis>-subunits

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic -subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKIN3, a novel protein related to AKIN subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (-GBD) identified in the mammalian AMPK1. In spite of its unusual features, AKIN3 complements the yeast sip1sip2gal83 mutant. Moreover, interactions between AKIN3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKIN3 is a -type subunit. A search for -type subunits revealed the existence of 3-type proteins in other plant species. Furthermore, we suggest that the AKIN3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKIN3-type subunits, involved in several functions among which some could be plant specific.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Structural and Functional Features of the Escherichia coli F 1 -ATPase

The structural organization and overall dimensions of the Escherichia coli F₁-ATPase in solutionhas been analyzed by synchroton X-ray scattering. Using an independent ab initio approach,the low-resolution shape of the hydrated enzyme was determined at 3.2 nm resolution. Theshape permitted unequivocal identification of the volume occupied by the _{3 3} complex ofthe atomic model of the ECF₁-ATPase. The position of the ^ and subunits were found byinteractive fitting of the solution scattering data and by cross-linking studies. Laser-inducedcovalent incorporation of 2-azido-ATP established a direct relationship between nucleotidebinding affinity and the different interactions between the stalk subunits and with the threecatalytic subunits () of the F₁-ATPase. Mutants of the ECF₁-ATPase with the introductionof Trp-for-Tyr replacement in the catalytic site of the complex made it possible to monitorthe activated state for ATP synthesis (ATP conformation) in which the and subunits arein close proximity to the subunits and the ADP conformation, with the stalk subunits arelinked to the subunit.     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a     

Identification of high molecular weight antigens structurally related to gamma-glutamyl transferase in epithelial tissues

Summary Heterologous antibodies to gamma-glutamyl transferase (GT), an ectoenzyme associated with the apical surface of many types of epithelial cells involved in secretion and transport, have been used to identify and partially characterize the spectrum of antigens in a series of epithelial tissues that exhibit a range of enzyme activities. In addition to antigens corresponding to the subunits of the active enzyme (mol wt 55K, 30K), antigens of mol wt 85–95K have been detected using an antibody raised against the enzyme purified in nonionic detergent. The latter species are shown to share antigenic determinants with and to be structurally related to the enzyme subunits; however, they do not bind significantly to antibodies raised to protease-solubilized GT. Further, they constitute the major antigens in tissues that exhibit relatively low levels of enzyme activity. These polypeptides are apparently larger than a recently characterized biosynthetic precursor of the GT subunits. Although they do not have GT activity themselves and their function is undefined, the possibility that they may represent highly glycosylated polypeptides related either to GT precursors (that persist without processing) or to the large enzyme subunit merits consideration.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Evolutionary relationship of the members of the sulphur-rich hordein family revealed by common antigenic determinants

Summary Five monoclonal antibodies raised against an enriched C hordein fraction have been characterized in detail and were found to be specific for the members of the sulphur-rich hordein family. Two antibodies specific for B hordein polypeptides were identified, one of which reacted predominantly with CNBr cleavage class III polypeptides. 1 hordein was recognized by two antibodies, of which one also reacted with 2 hordein and several members of the CNBr cleavage class II B hordein polypeptides. One antibody recognized 3 hordein but cross-reacted at higher antibody concentration with almost all of the B and C hordein polypeptides. The specificity of the monoclonal antibodies was confirmed by Western blotting of one- or two-dimensionally separated hordein from the B hordein-deficient mutant hor2ca and its wild-type Carlsberg II and the 3 hordein-deficient genotype Nevsky. The identification of the hordein-specific monoclonal antibodies was further supported by immune precipitation of in-vitro transcribed and translated 2 hordein, and hor2ca and Carlsberg II mRNA translation products. The monoclonal antibodies were used to screen for mutants in hordein synthesis. Two mutants, one deficient in 1 hordein synthesis and a second in 2 or closely related B hordein polypeptides were identified. A model is proposed for the evolution of the sulphur-rich hordein loci Hor5 and Hor2.     

Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.