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.     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation

Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles.     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

Action of Some Analogs of Nitrosoguanidine on the Chloroplasts of Euglena gracilis

SYNOPSIS. Several analogs of N-methyl-N'-nitro-N-nitrosoguanidine (NG) share with it the property of rendering Euglena permanently unable to form chloroplasts (i.e., of bleaching Euglena ). All the active compounds contain an N-alkyl-N-nitrosamine group. Analogs in which the nitroso group is replaced by a nitro group or by a hydrogen atom do not bleach. The possible mode of action of alkylnitrosamines is discussed.     

Large-Scale Autotrophic Culture of Euglena gracilis*

SYNOPSIS. Large quantities of autotrophic Euglena gracilis can be obtained in a short time using simple and inexpensive equipment. Cultures grown in flasks on a rotary shaker are transferred to a polyethylene drum containing 180 liters of medium. The drum, which is illuminated with both internal and external fluorescent lamps, contains a coil of vinyl tubing carrying tap water for temperature control and 2 vinyl tubes for air and CO₂. Sterilization of the drum or the medium was unnecessary, but can be accomplished by gassing the drum with ethylene oxide or overnight exposure to germicidal lamps and by dispensing the medium into the drum through a large Millipore apparatus. Yields of 289–330 g of fresh weight material have been obtained after one week's growth. Shorter growth periods yielded 134–160 g of fresh weight material. Ten g of purified chloroplasts have been obtained from 160 g of cells grown in this manner.     

Properties of Photosynthetic Mutants Isolated from Euglena gracilis

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

Chemosensory Responses Toward Oxygen in Euglena gracilis*

SYNOPSIS Euglena gracilis strain Z, at a concentration of 10⁶ cells/ml and in containers of ∽ 0.1-mm thickness, spontaneously forms dynamic ring patterns in the dark. These patterns are modified differentially by illumination with red and with blue light. The red light effect is abolished by treatment with an inhibitor of photosynthesis. Pattern formation is apparently the result of chemophobic responses to oxygen dissolved in the medium. Euglena can respond to both negative and positive concentration gradients, depending upon the absolute magnitude of oxygen concentration. The photo- and chemosensory transduction systems of Euglena interact at a stage which precedes the overt expression of motor responses.     

Factors affecting division rhythmicity in Euglena gracilis

In phototrophic culture of Euglena gracilis, good synchrony was found only under rather restricted programs of light-dark cycles, and rather narrow ranges of temperature and light intensity, when cultures were flushed with air fortified with adequate amounts of CO₂. When flushed with air alone, CO₂ was found to be limiting, and while cell divisions were rhythmic, less than a doubling of cell number occurred in division bursts. With air as gas phase, rhythmic division activity was maintained over wide ranges of temperature, light intensity, and the ratio of light:dark in a given program; all these factors affected the amplitude of the division burst, however.     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Light Activation of Mg-dependent Adenosine 5'-Triphosphatase in Isolated Euglena Chloroplasts

Enhancement of Mg²⁺-dependent ATPase activity in Euglena gracilis chloroplasts by light in the presence of a sulfhydryl compound has been demonstrated. A number of uncouplers and energy transfer inhibitors were studied for their effects on the light enhancement of ATPase activity simultaneously with their effects on photophosphorylation. Results suggest that the light-enhanced ATPase activity in Euglena chloroplasts is an energy-initiated process and that the energy is supplied through electron flow upon illumination of the chloroplasts. However, by studying the difference in their response toward the various uncouplers and inhibitors, it seems that the two processes (photohydrolysis of ATP and photophosphorylation) share only the latter part of their energy-transferring pathway.     

Glucose Utilization by Euglena gracilis var. bacillaris at Higher pH*

SYNOPSIS. Euglena gracilis var. bacillaris is able to grow luxuriantly on glucose in a mineral salts medium at pH 6.8–7.1 following an adaptation period of about 200 hr. If adapted cells are used as an inoculum or if 0.1% glycine is included in the medium, the lag is shortened to 70–100 hr. Inclusion of 0.1% acetate in the medium produces a diphasic growth pattern, with acetate being metabolized first, followed by the later (about 400 hr) utilization of the glucose. Glucose utilization was found to be sensitive to pH as compared to growth on ethyl alcohol. However, glycine partially overcame this sensitivity. Glycine is maximally stimulatory with regard to growth on glucose at pH 7.0 at a concentration of 0.03%, thus suggesting that it functions as a sparking substance. Glycine markedly stimulates the assimilation of ¹⁴C-glucose. A number of Krebs cycle acids and amino acids were also found to stimulate ¹⁴C-glucose assimilation at neutral pH. Adaptation to glucose utilization at neutral pH was due to the appearance of mutants able to grow more rapidly under these conditions. The nature of this mutation was not determined.     

A Study of Plastoquinones in Photochemical Reactions in the Chloroplasts of Euglena gracilis Strain Z

Plastoquinone-9 (PQ-9) was isolated from the chloroplasts of Euglena gracilis Strain Z and spinach. The functional involvement and the structural specificity of PQ-9 in photochemical reactions was investigated in the isolated chloroplasts of Euglena gracilis. It was found that PQ-9 was required for both photoreduction of ferricyanide and photosynthetic phosphorylation in Euglena chloroplasts. The structural integrity of PQ-9 was not required to the same degree in the 2 photochemical reactions. Photosynthetic phosphorylation seemed to require the entire molecular structure of PQ-9 for the activity, whereas shortening in isoprenoid chain and modification of quinoid nucleus of PQ-9 do not seem to alter the photoreduction activity significantly. Addition of PQ-9 to the lyophilized Euglena chloroplasts inhibited the photoreduction of ferricyanide significantly, while it stimulated photosynthetic phosphorylation activity.     

Biosynthesis of Phosphatidylcholine in Euglena gracilis*†

SYNOPSIS. Extracts of Euglena gracilis carry out a very rapid but limited synthesis of phosphatidylcholine when S-adenosylmethionine or ATP and methionine are supplied. Cytidinediphosphocholine apparently is not utilized. Qualitatively the same results are obtained whether the cells are light- or dark-grown.