Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

Ontogenetic Change of the PSI- and PSII-Distribution in the Thylakoid System of Euglena gracilis

The thylakoid membranes of isolated Euglena chloroplasts wereseparated into two fractions by aqueous two-phase-partitioning(mixture of dextran 500 and poly(ethylene glycol) 4000) followingpress disruption. These two fractions differ in many respectsduring most of the cell cycle of this alga in comparison withthe thylakoid characteristics of higher plants or green algae.The amount of thylakoid membranes with separation characteristicscomparable with inside-out-vesicles of higher plant chloroplastschanges depending on the cell cycle stage of Euglena gracilis.Photosystems II and I are not restricted to one fraction. Boththylakoid membrane fractions evolve oxygen photosynthetically.When chloroplast differentiation in Euglena gracilis is complete(i.e. at the end of the light-time) the composition and thephotosynthetic efficiency of the two thylakoid fractions aregenerally equal. Photosystem I-related LHCI is present in bothfractions. Photosystem II-related CP29, however, was only detectedin unfractionated thylakoid membranes. The implications forthylakoid organization in Euglena chloroplasts are discussed. Key words: Euglena gracilis, photosystem I, photosystem II, stacking, thylakoids     

Carotenoid composition of spinach chloroplast grana and stroma lamellae

Stroma lamellae and grana stacks prepared by French press rupture of spinach (Spinacia oleracea) chloroplasts contain similar amounts of β-carotene on a protein basis. The grana fraction has considerably more xanthophylls than does the stroma fraction. Total carotenoid to chlorophyll ratios are similar for both fractions.     

ULTRASTRUCTURE OF THE THERMOPHILIC BLUE-GREEN ALGA,SYNECHOCOCCUS LIVIDUS COPELAND1

The ultrastructure of Synechococcus lividus Copeland, a thermophilic blue-green alga, was studied in thin sections. The cell envelope reveals striking similarities with that of some gram-negative bacteria. In contrast to bacteria and to many other species of blue-green algae, ribosomes are predominantly found in the central nuclear region and appear to be associated with the DNA fibrils. Thylakoids (photo-synthetic lamellae) are arranged as concentric shells, around the nuclear equivalent, lying nearly parallel to one another and to the plasma membrane. Both plasma and thylakoidal membranes, as described by other authors for different Cyanophyceae, are of the unit membrane dimension and morphology. Various types of intracellular inclusions are found: (1) Lipid inclusions, located in the cytoplasm are similar to the osmiophilic globules of higher plant chloroplasts. (2) Polyphosphate inclusions (or volutin) resembling those of other species are generally found at the cell poles but within the nuclear region. (3) Polyhedral inclusions also located in the nuclear region are clearly recognized to be different from the polyphosphate bodies, but their function remains unknown.     

On the localization of polyribosomes in the system of chloroplast lamellae

From chloroplasts of 10-day-old pea seedlings exposed to the light for 19 h, two fractions have been isolated. One of them is rich in lamellae of the stroma, and the other is rich in thylakoids and fragments of the grana. These fractions have been obtained after centrifugation of chloroplasts disrupted by osmotic shock in a discontinuous sucrose gradient. The fraction containing thylakoids of grana differs from the fraction of lamellae of the stroma in its higher content of RNA and DNA as related to protein and in the capacity to incorporate intensively ¹⁴C amino acids into proteins. After its treatment with detergents (0.5% sodium deoxycholate and 0.4% Triton X-100) and repeated centrifugation in the discontinuous sucrose gradient it dissociates further into two fractions. During electron microscopic studies one of these fractions displays partially disrupted grana and the other exhibits extensive networks of polyribosomes incompletely liberated from proteins, including the de novo synthesized protein.The similar treatment of the fraction rich in lamellae of the stroma does not result in the liberation of polyribosomes.It is concluded that in this stage of chloroplast development, polyribosomes occurring in the lamellae system are localized in the thylakoids of grana and are not bound to lamellae of the stroma.     

Free flow electrophoresis of chloroplasts

Highly purified intact chloroplasts were isolated from spinach (Spinacia oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO₂ fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lamellae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities.     

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity.The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique.Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks.The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.     

Purification and species distribution of rubisco activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

Reunification of sub-cellular fractions of bryopsis into viable cells

Protoplasm from Bryopsis maxima, a coenocytic green alga, was dissociated into two fractions; chloroplasts and a protoplasmic fraction without chloroplasts (PF). PF induced nuclei, mitochondria, dictyosome, endplasmic reticulum, etc. These two fractions were reunited and protoplasts with complete plasmamembrane were reformed within 10 h. Cell wall regeneration was observed 24–30 h after reuniting. New cells began budding 2–3 days after, and 1 month after reunification, they had developed into mature plants.     

A study of several blue-green algae in the electron microscope

Summary All of the three blue-green algae, Anabaena cylindrica, Mastigocladus laminosus and Nostoc muscorum are characterized by the presence of multi-layered envelopes (sheath, wall and plasma membrane), photosynthetic lamellae and a variety of intracellular granules. Sections of heterocysts of Anabaena cylindrica showed the presence of an internal membrane system as well as lamellae. An unusual feature of the structure of Nostoc muscorum was the presence of densely stained intracellular membranes or lamellae. The results emphasize the variability in appearance of the internal structure of the blue-green algae and point to the need for detailed investigations of the influence of change in physiological environment on the anatomy of these organisms.     

Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP⁺ or ferricyanide. P/2e⁻ ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.

Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C₅₅₄ and C₅₄₉ and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C₅₅₄ or plastocyanin, and may be a coupling factor.

Analysis of the well-washed membranes by low temperature (77°K) difference spectrophotometry reveals the presence of cytochrome b₆ and a bound form of cytochrome C₅₅₄ in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C₅₅₄, relative to cytochrome b₆ is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C₅₅₄, which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b₆: chlorophyll a was found to be 1:500.

Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C₅₄₉.

     

Quantification of mRNA in Chloroplasts of Chlamydomonas reinhardii: Equal Distribution of mRNA for a Soluble and a Membrane Polypeptide in Stroma and Thylakoids

The relative contents of the mRNAs were analyzed for the 32kDa herbicide-binding protein and for the large subunit of ribulose-l,5-bisphosphatecarboxylase in the membrane fraction and in the soluble fractionof chloroplasts from Chlamydomonas reinhardii. The presenceof mRNA for the two proteins in both subchloroplast fractionswas demonstrated by in vitro translation of isolated RNA inthe reticulocyte lysate. The relative amounts of the two mRNAswere measured by hybridizations with cloned chloroplast DNAprobes at two stages of the cell cycle. Both mRNAs were distributedin the same ratio between membrane and soluble fractions, about75% of both mRNAs being in the membrane and 25% in the solublefraction. Therefore, in chloroplasts the accumulation of mRNAson thylakoid membranes does not reflect the final localizationof soluble and membrane proteins. ¹Present address: Department of Biology, Ben Gurion University,Beer-Sheva, Israel. (Received April 28, 1987; Accepted September 29, 1987)     

Purity of Chloroplasts Prepared from the Siphonous Green Alga, Caulerpa simpliciuscula, as Determined by Their Ultrastructure and Their Enzymic Content

The ultrastructure and enzyme distribution in chloroplasts and other subcellular fractions isolated from the siphonous green alga, Caulerpa simpliciuscula, are described. The isolated chloroplasts were similar in appearance to those in the tissue from which they were derived, and in typical preparations 70% or more were intact. Chloroplasts which had lost their outer envelopes could be separated from intact plastids by centrifugation at low speeds through gradients of colloidal silica. Intact chloroplasts separated in this way retained their photosynthetic capacity and were impermeable to ferricyanide ions. The chloroplast preparations separated by differential centrifugation and refractionated using either discontinuous or continuous Percoll gradients contained non-chloroplast material. It was estimated that this amounted to a maximum of 10% of the mitochondrial population and 6% of cytoplasm extracted from the plant. The contaminating material surrounded the chloroplasts in a thin layer and was surrounded by a membrane.     

Effect of Chloramphenicol on Formation of Chloroplast Structure and Protein During Greening of Etiolated Leaves of Phaseolus vulgaris

Greening of leaves of Phaseolus vulgaris in the presence of chloramphenicol inhibits formation of A) total chloroplast protein, B) an easily extractable fraction removed during isolation of chloroplasts in isotonic media by differential centrifugation, and C) the insoluble lamellar fraction which remains after extracting osmotically shocked freeze-dried plastids. The inhibition of insoluble chloroplast protein formation is correlated with decreased formation of lamellae and increased formation of vesicular structures. In contrast, chloramphenicol increases the formation of a fraction not removed during differential centrifugation, but removed by water extraction after osmotic shock and freeze-drying of chloroplasts. Analysis of this fraction by electrophoresis and column chromatography, indicates that the increased accumulation of this protein fraction is largely due to accumulation of a protein which is normally present in this fraction in small quantities. It was suggested that this protein may be a precursor which is normally incorporated into the lamellae. The protein extracted from freeze-dried chloroplasts of chloramphenicol treated chloroplasts contains a smaller proportion of one or more proteins than a similar extract of untreated plastids. However, per plastid, no such difference exists.     

Relationships between the Transition of the Physical Phase of Membrane Lipids and Photosynthetic Parameters in Anacystis nidulans and Lettuce and Spinach Chloroplasts

The transition of the physical phase of lipids in membrane fragments of a blue-green alga Anacystis nidulans was studied by a spin labeling technique. The maximum hyperfine splitting of the electron spin resonance spectrum of the N-oxyl-4′, 4′-dimethyloxazolidine derivative of 5-ketostearic acid plotted against the reciprocal of the absolute temperature gave a discontinuity point that was characteristic of a transition of the physical phase of the hydrocarbon region of membrane lipids. The phase transition appeared at approximately 13 or 24 C in the organisms grown at 28 or 38 C, respectively.     

Use of streptavidin to detect biotin-containing proteins in plants

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.