Localization and Orientation of Subunit Delta of Spinach Chloroplast ATP-Synthase within the CF0CF1 Complex. 1. Distinction of Shielded and Exposed Surfaces of Delta on the Thylakoid Membrane
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A new polyclonal antiserum against spinach CF1 subunit delta was produced in rabbits. It decorates only one band at 21 kDa in Western immunoblots of thylakoid proteins and does not react in ELISA with delta-free four subunit CF1(-delta); therefore it is regarded monospecific. The polypeptide used as immunogen had been purified by HPLC. Earlier antisera against CF1 delta cross-react with CF1 subunit beta. The new antiserum 306 contains different antibodies; some can be absorbed with thylakoids, i.e. by delta within the assembled CF0CF1 complex on the membrane, others still react in ELISA with isolated CF1. The former antibodies agglutinate thylakoids and inhibit PMS cyclic photophosphorylation. Therefore we conclude that part of the surface of CF1 subunit delta is exposed within the quaternary structure of the ATP-synthase complex of photosynthetically active thylakoids, but part of the surface of delta is shielded. Trypsination of isolated delta occurs at several sites, but this protease does not attack delta in situ, nor does aminopeptidase. Staphylococcus aureus protease V8 digests CF1 delta after isolation at residues Asp53, Glu61, Glu95 and Glu106, but has no access to these residues of delta in situ. Thus CF1 subunit delta seems to be shielded within the CF0CF1 complex to a large degree. Direct agglutination of thylakoids by anti delta serum 306 was weak and could be improved tenfold by a Coombs serum (goat anti rabbit gammaglobulin), whereas an anti beta serum agglutinated directly. From this we conclude that delta is not accessible at the top of the enzyme; the exposed part is extending below the large subunits alpha and beta and oriented towards the membrane.Keywords:
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Nuclear gene
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Digitonin
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Chenopodiaceae
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An intrathylakoid electron opaque substance, further referred to as loculin, is found in 80-90 % of thylakoids of tansy leaf mesophyll chloroplasts at the stage of flower bud formation and flowering. Upon conventional isolation of chloroplasts in aqueous solution, and fixation in osmium solution alone, loculin is not retained in thylakoids. Preliminary fixation of leaves in glutaraldehyde makes it possible to isolate chloroplasts without injuring the envelope and stroma (glutar chloroplasts), and loculin is retained in thylakoids under these conditions. Upon prolonged incubation of glutar chloroplasts (for 24 h), loculin leaves thylakoids in the form of drops concentrating on the chloroplast envelope. Upon crossing the thylakoid membrane and chloroplast, loculin properties remain unchanged. It is assumed that loculin is an important metabolite necessary for active growth.
Chloroplast membrane
Glutaraldehyde
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Polysome
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Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.
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Abstract Bienertia sinuspersici is a single-cell C4 plant species of which chlorenchyma cells have two distinct groups of chloroplasts spatially segregated in the cytoplasm. The central vacuole encloses most chloroplasts at the cell center and confines the rest of the chloroplasts near the plasma membrane. Young chlorenchyma cells, however, do not have large vacuoles and their chloroplasts are homogenous. Therefore, maturing Bienertia chlorenchyma cells provide a unique opportunity to investigate chloroplast proliferation in the central cluster and the remodeling of chloroplasts that have been displaced by the vacuole to the cell periphery. Chloroplast numbers and sizes increased, more notably, during later stages of maturation than the early stages. Electron tomography analyses indicated that chloroplast enlargement is sustained by thylakoid growth and that invaginations from the inner envelope membrane contributed to thylakoid assembly. Grana stacks acquired more layers, differentiating them from stroma thylakoids as central chloroplasts matured. In peripheral chloroplasts, however, grana stacks stretched out to a degree that the distinction between grana stacks and stroma thylakoids was obscured. In central chloroplasts undergoing division, thylakoids inside the cleavage furrow were kinked and severed. Grana stacks in the division zone were disrupted, and large complexes in their membranes were dislocated, suggesting the existence of a thylakoid fission machinery.
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In order to determine the effect of senescence on the structure and function of chloroplasts in leaf mesophyll cells of eggplant(Solanum melongena Linn.),chloroplasts in immature and senescing leaves were comparatively studied by means of electron microscopy.Chloroplasts in the young leaf mesophyll cells contain cylindrical grana that are connected by stroma thylakoids,rich starch grains,and a few small plastoglobuli that are less than 0.1 μm in diameter.The thylakoids are stained more strongly.However,in the chloroplasts in the old leaves,grana become spherical,thylakoids are stained weakly,stroma thylakoids become less in number,starch grains are reduced or disappeared,plastoglobuli increase in number and size with diameter up to 0.9 μm,and phenolic accumulation appears.It is suggested that leaf senescence of eggplant induces change to thylakoid membrane constituents,inhibits starch accumulation,promotes plastoglobule accumulation,and stimulates phenolic accumulation.
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Objective:To identify the immune characteristics elicited by immunogen prepared in two different ways a gainst a 36AApeptide.Methods:Rabbits were immunized by a 36AAhapten coupled with carrier proteins or recombinant fusion protein separately.The kinetics and specificities of antibody responses were compared.Results:After about 3 months of primary immunity, the antisera elicited by two kinds of immunogen reached the peak titer.The highest titer elicited by the im munogen prepared with coupledhapten method reached 1∶12 000, while the highest titer elicited by the immunogen prepared with gene engineering method was 1∶6 000.But the immune response produced by the recombinant fusion protein was more longlasting.Conclusion:Both kinds of immunogen can successfully elicit 36AA specific antibody response in rabbits, which have different immune characteristics.To couple the hapten with vector proteins is a quicker and more effective way to prepare the immunogen.
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