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.     

A Photorepressible Isozyme o Malic Enzyme in Euglena gracilis Strain Z*†

SYNOPSIS. Isozymes of malic enzyme in Euglena gracilis strain Z were analyzed by starch-gel electrophoresis. Wild-type and heat-bleached strains were cultured in the light and the dark in the presence of various carbon sources. An isozyme detectable in heterotrophic cultures was repressed by photosynthesis. A model is proposed to explain photorepression of this isozyme.     

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.     

Mono-ADP-Ribosylation of Arginine Residue of Euglena gracilis Z in Synchronous Culture

ABSTRACT. In Euglena gracilis Z, a considerably high activity of mono-ADP-ribosyltransferase occurred and change of it was accompanied by a cell cycle induced by a light-dark cycle. The enzyme activity was strongly inhibited by L-arginine and supported in the presence of poly-L-arginine as a substrate, indicating that ADP-ribosylated amino acid is an arginine residue. Arginine: mono-ADP-ribosyltransferase activity was found in the chloroplasts, mitochondria, microsomes and cytosol as judged from marker enzyme activities and the activity in each organelle fluctuated with the cell cycle.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

The Molecular Biology of Euglena gracilis. X. Amino Acid Composition of Protein

SYNOPSIS. Cells of Euglena gracilis strain Z were extracted with trichloroacetic acid. Samples of gross cellular protein were hydrolyzed by a variety of reagents. Amino acids released by these procedures were analyzed and the overall composition of cell protein was quantitatively determined.     

The Relationship of Fixed Carbon and Nitrogen Sources to the Greening Process in Euglena gracilis strain Z*

SYNOPSIS The pattern of chloroplast development was followed in Euglena gracilis strain Z greening in media with a variety of fixed carbon and nitrogen sources. The greening pattern of cells grown in inorganic medium with added ethanol or glucose involves an inhibition of chloroplast development when compared to that of cells grown in inorganic medium alone. Several nitrogen sources were tested to ascertain their effectiveness in relieving the inhibition of chloroplast development by glucose. Of those, only 0.05% (w/v) (NH₄)₂ SO₄ accelerated the recovery from the inhibition after most of the glucose had been removed from the medium by the cells. The other nitrogen sources tested were not effective. An inhibition of chloroplast development, similar to that observed in cells greening in the presence of glucose, was seen in cells greening in an ethanol-containing medium. These cells, however, had a different response upon the addition of 0.05% (NH₄)₂ SO₄. They appeared to recover from the inhibition of chloroplast development, even before the ethanol was removed from the medium by the cells. A slight enhancement of chloroplast development was noted in cells greening in an inorganic medium with glycine or serine. Other amino acids tested had little or no effect.     

High Speed Cinemicrography of the Direct Photophobic Response of Euglena and the Mechanism of Negative Phototaxis*

SYNOPSIS. The shock reaction of Euglena gracilis strain Z to a sudden increase in light intensity (the “direct photophobic response”) was examined by high speed cinemicrography. The response is expressed as a turning reaction toward the dorsal side of the cell, after a transduction time of 0.1–0.5 sec after the onset of stimulation. Transduction times, turning rates, and flagellar beat frequencies were measured by analyzing the filmed sequences. The experimental data are consistent with a mechanism of directional homeostasis in negative phototaxis that is based upon shading of the photoreceptor by the cell's posterior end.     

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.     

Nitrofuran-Reducing Enzymes of Euglena*

SYNOPSIS. Euglena gracilis strain Z, green, dark-grown, and “bleached”with N-methyl-N-nitro-N-nitrosoguanidine, was found to contain 2 soluble enzymes which reduce nitrofurans. A small amount of activity was demonstrated also in a particulate fraction of sonic extracts, but none in isolated chloroplasts. The reduction of 5 nitrofurans, having widely differing bleaching activities, by each of the 2 enzymes was examined.     

Motor Responses to Polarized Light and Gravity Sensing in Euglena gracilis*†

SYNOPSIS. Euglena gracilis strain Z has a motor response which results in orientation with respect to the polarization of a light stimulus. Cells swim preferentially in a direction perpendicular to the plane of polarization of the stimulus. If 2 polarized stimuli are given from opposite directions, the preferred direction is, under certain circumstances, at right angles to the directions of both stimuli. Euglena also preferentially assumes an orientation that is at right angles to the force of gravity. The relationships between these responses and phototactic movements oriented with respect to the direction of the stimulus are discussed.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Aldolase Levels in Wild-Type and Mutant Euglena gracilis

SYNOPSIS. Wild-type Euglena gracilis var. baciliaris , strains B and Z, synthesize 2 distinct fructose-1,6-diphosphate aldolases. Amounts of the 2 activities depend upon conditions of growth. The class I enzyme, with activity similar to that found in various photosynthetic tissues, is formed during regreening of dark-grown cultures incubated in the light. Very low activity of the class I enzyme is also found in mutant strains W₃BUL and W₈BHL, both of which apparently lack plastid DNA. The significance of these findings is discussed.     

The natural herbicide, cyanobacterin, specifically disrupts thylakoid membrane structure in Euglena gracilis strain Z

Abstract Cyanobacterin is a natural product produced by the cyanobacterium (blue-green alga), Scytonema hofmanni . The compound has been chemically characterized and shown to inhibit electron transprot in photosystem II. Although the herbicide is lethal to photoautotrophs, photoheterotrophically-grown organisms such as Euglena gracilis can survive and grown in saturating concentrations of cyanobacterin. Electron micrographs of treated E. gracilis cells show extensive damage to the thylakoid membranes of the chloroplasts, similar to the effects observed with 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU). Unlike the synthetic herbicide, cyanobacterin specifically disrupts thylakoid membranes and does not affect other cellular membranes or heterotrophic growth.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Influence of Culture pH on Chloroplast Structure in Euglena gracilis

Chloroplasts of Euglena gracilis grown with phototrophic nutrition at pH 3.0 were compact, while those in cells grown at pH 8.1 were swollen with widely separated lamellae.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

ATPase Activity in Flagella from Euglena gracilis. Localization of the Enzyme and Effects of Detergents*

SYNOPSIS. The biochemical effects of some detergents on the ATPase activity of isolated flagella from Euglena gracilis are related to morphologic obliterations induced by those detergents. Enzymic activity can be localized by electron microscopy along the microtubules and also on the paraflagellar rod. The nonionic detergent digitonin solubilizes the enzyme linked to dyneinic arms, whereas the activity linked to residual structures appears enhanced. These results support the hypothesis that the paraflagellar rod may be a structure actively related to the motility of this type of flagellum.     

The Molecular Biology of Euglena gracilis. VIII. Glycitols and Monosaccharides

Mannitol is identified as the major component of the small molecule pool of Euglena gracilis strain Z. Smaller amounts of glycerol also are found. Results of tracer studies and enzymic measurements indicate that both major mannitol pathways found in bacteria are present in Euglena.     

Light-Induced Body Movement of Euglena gracilis Coupled to Flagellar Photophobic Responses by Mechanical Stimulation*†

SYNOPSIS. In low viscosity media, Euglena gracilis strain Z responds to a sudden change in light intensity by a cessation of forward movement, followed by a reorientation of the locomotor flagellum which results in turning of the cell around the lateral axis (photophobic response). At a viscosity interface between low [～ 1 cP (centipoise)] and high (4000 cP) media, the cells exhibit avoidance responses or become immobilized in the higher viscosity medium. Upon changing the light intensity, free swimming cells have photophobic responses, while immobilized ones undergo body contractions. For cells immersed in media of varying viscosity, the delay between light stimulation and body contraction (transduction time) is shortest at high viscosities. From 500 to 2000 cP, where the cells are capable of both movement and light-induced body contractions, there is a logarithmic dependence of the transduction time on the viscosity. The transduction time does not vary appreciably with the intensity of the primary light stimulus within a range of 0.14-1.13 kW/m².