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.     

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.     

Subcellular Localization of the GABA-Shunt Enzymes in Euglena gracilis Strain Z

SYNOPSIS. Glutamate decarboxylase, γ-aminobutyrate-α-ketoglutarate aminotransferase and NAD-linked and NADP-linked succinic semialdehyde dehydrogenase, all constituting the GABA (γ-aminobutyrate)-shunt pathway of glutamate metabolism are localized in the mitochondrial matrix in a streptomycin-bleached mutant of Euglena gracilis strain Z. Glutamate dehydrogenase, requiring NADP as the cofactor, was distributed in the cytoplasm. An improved version of the controlled digestion method for preparing Euglena mitochondria, which involves use of trypsin and a trypsin inhibitor and removal of broken cells before mechanical disruption of cells, is also described.     

Regulation by Ammonium of Variation in Heterotrophic CO2 Fixation by Euglena gracilis During Growth Cycles*

SYNOPSIS Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH₄* from the medium during growth. It is demonstrated that exogenous NH₄⁺ regulates a component of heterotrophic CO₂ fixation and that another component is independent of NH₄⁺. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO₂, light) and for a permanently bleached strain (E. gracilis SB3). Some kinetics of the NH₄⁺ regulation are presented.     

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.     

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.     

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.     

Circadian Rhythm Changes in Autotrophic Euglena Induced by Organic Carbon Sources*

SYNOPSIS. Acetate added to autotrophic Euglena cultures changed the period length of the circadian rhythm of phototaxis. Phase shifts were induced by acetate pulses. Since transition from one metabolic state to another (autotrophic/mixotrophic) caused a phase shift or a period change, such effects possibly result from switching metabolic pathways. As suggested (Brinkmann, K., 1966. Planta 70 , 344–89), differences in the temperature responses of the rhythm in mixotrophic and autotrophic cells might also be caused by participation of different metabolic pathways with different Q¹⁰ values, e.g. dark reactions vs photochemical reactions. However the Q¹⁰ of a given dark reaction, e.g. protein synthesis, can differ in the 2 states. Therefore temperature experiments alone do not suffice for deciding whether the pathways include photochemical reactions, dark reactions, or both.     

In vivo and in vitro methylation of the elongation factor EF-Tu from Euglena gracilis chloroplast

Based on amino acid sequence similarities between the methylated elongation factor EF-Tu from Escherichia coli and the EF-Tu from Euglena gracilis chloroplast, we predicted that the latter could also be methylated in the presence of an appropriate methyltransferase. We found that, as reported for the eubacterial homologous protein, the organellar factor could be methylated in vivo and in vitro to yield monomethyllysine.     

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.     

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.     

Comparison of the potencies of (+)- and (−)-2-ethylhexanoic acid in causing peroxisome proliferation and related biological effects in mouse liver

Male C57BL/6 mice were exposed to 1% (w/w) (+)- or (?)-2-ethylhexanoic acid or an equimolar mixture of these enantiomers in their diet for 4 or 10 days. A significant increase in liver weight and a 2- to 3-fold increase in the protein content of the mitochondrial fraction were seen in all cases. Peroxisomal palmitoyl-CoA oxidation was increased 2- to 3.5-fold after 4 days of treatment and 4- to 5-fold after 10 days, while the corresponding increases in peroxisomal lauroyl-CoA oxidase activity were 2- to 3-fold and 9- to 12-fold, respectively. Peroxisomal catalase activity was unchanged, whereas the microsomal and cytosolic activities were increased 2- to 3-fold and 6- to 16-fold, respectively. These treatments also induced microsomal ω-hydroxylation of lauric acid 7-fold and soluble epoxide hydrolase activity in the mitochondrial and cytosolic fractions, as well as microsomal epoxide hydrolase activity about 50–100%. The only significant differences observed between the effects of (+)-2-ethylhexanoic acid and its (?)-enantiomer were on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity after 4 days of treatment. In both these cases the (+)-enantiomer resulted in increases which were 50–75% greater than those seen with the (?)-form. © 1994 Wiley-Liss, Inc.     

Influence of the alternative respiratory pathway on the adenylate pools in heterotrophic Euglena gracilis

Etiolated Euglena gracilis Pringsheim, strain Z, were cultured in a lactate medium either in the presence of 2 μ M antimycin A for cells adapted to this inhibitor, or in the absence of antimycin A for controls. The adenylates (ATP, ADP and AMP) and the energy charge (EC) were followed during the growth of both types of cells. The effects of KCN, salicylhydroxamic acid (SHAM) and rotenone on the respiration and the adenylate pool, were investigated during the exponental and stationary phases. EC values of controls and antimycin-adapted cells were not significantly different during culture. In the logarithmic phase, EC of controls was unaffected by 3 m M SHAM, an inhibitor of the alternative pathway, but markedly decreased by 0.3 m M KCN, which inhibits the cytochrome pathway. In contrast, in antimycin-adapted Euglena , in which the cytochrome pathway was blocked, ATP content and EC were markedly lowered in the presence of SHAM but slightly increased by 0.3 m M KCN. The combination of the preceeding treatments, as well as 15 m M KCN alone, were deleterious for both types of cells, in the logarithmic and the late stationary phases. The data indicate that the energy level in Euglena was dependent on the alternative pathway when the cytochrome pathway was blocked. Such dependence could be explained by the engagement of the first rotenone-sensitive site of phosphorylation. Indeed, 50 μ M rotenone caused a similar relative decrease of oxygen consumption and ATP content in controls and in antimycin-adapted Euglena . In the absence of cytochrome respiration, the alternative pathway allowed electrons to flow through this first segment of the respiratory chain, and ATP production by the first site of phosphorylation.     

Coexistence of β1- and β2-Adrenoceptors in the Melanophore of the Goby Tridentiger obscurus*

The effects of β-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these β-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α-and β-adrenoceptors, and that the activation of the β-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two β₁-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β₁-receptors. By contrast, the pigment-dispersing effects of two β₂-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β₁- and β₂-adrenoceptors in the melanophores. The relative numbers of β₁- and β₂-adrenoreceptors as a percentage of the total population of β-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Peroxisomes in the Alga Vaucheria are Neither of the Leaf Peroxisomal Nor of the Glyoxysomal Type*,1

Microbodies were isolated from the freshwater alga Vaucheria sessilis as well as from a marine Vaucheria. The organelles equilibrated on sucrose gradients at densities 1.23 g . cm^?3 and 1.24g . cm^?3, respectively. On electron micrographs they showed an ovoid or spheroid shape with a diameter of 0.5 to 0.8 μm. Besides catalase, the peroxisomes of both algae possess glycolate oxidase and glutamate-glyoxylate aminotransferase, but no other leaf-peroxisomal enzymes. Instead, the enzymes malate synthase and isocitrate lyase, which are markers of glyoxysomes in higher plants, are constituents of the peroxisomes in the marine as well as in the freshwater alga. Citrate synthase, aconitase, malate dehydrogenase and enzymes of the fatty acid β-oxidation pathway are located exclusively in the mitochondria. Therefore, the peroxisomes from Vaucheria do not belong to either the type of leaf peroxisomes or to the type of glyoxysomes.     

Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism

Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation.