Physiological characterization of gravitaxis in Euglena gracilis

The motile, unicellular freshwater flagellate Euglena gracilis uses external stimuli, like gravity, light or oxygen pressure in order to orient itself in its natural habitat. In the darkness the cells normally show a negative gravitactic behavior, that means they swim upward in the water column, Many ground and space experiment revealed that gravitaxis is most likely based on active physiological mechanisms (involvement of calcium, cAMP, membrane potential and other parameters).     

Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 × 10⁻¹¹ molar, while 7.4 × 10⁻¹⁰ molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes.     

Physiological role of rhodoquinone in Euglena gracilis mitochondria

Rhodoquinone (RQ) participates in fumarate reduction under anaerobiosis in some bacteria and some primitive eukaryotes. Euglena gracilis, a facultative anaerobic protist, also possesses significant rhodoquinone-9 (RQ9) content. Growth under low oxygen concentration induced a decrease in cytochromes and ubiquinone-9 (UQ9) content, while RQ9 and fumarate reductase (FR) activity increased. However, in cells cultured under aerobic conditions, a relatively high RQ9 content was also attained together with significant FR activity. In addition, RQ9 purified from E. gracilis mitochondria was able to trigger the activities of cytochrome bc1 complex, bc1-like alternative component and alternative oxidase, although with lower efficiency (higher Km, lower Vm) than UQ9. Moreover, purified E. gracilis mitochondrial NAD+-independent D-lactate dehydrogenase (D-iLDH) showed preference for RQ9 as electron acceptor, whereas L-iLDH and succinate dehydrogenase preferred UQ9. These results indicated a physiological role for RQ9 under aerobiosis and microaerophilia in E. gracilis mitochondria, in which RQ9 mediates electron transfer between D-iLDH and other respiratory chain components, including FR.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Heteroxanthin in Euglena gracilis

Summary 1. From a large scale preparation of Euglena gracilis, strain Z, besides the acetylenic pigments diatoxanthin and diadinoxanthin and the allene neoxanthin, an additional acetylenic xanthophyll has been isolated. 2. Mass and IR spectra and chemical reactions showed typical patterns of heteroxanthin from Vaucheria. 3. The pigment was transformed into diadinochrome-isomers with acidified acetone. 4. A partial synthesis of heteroxanthin from diadinoxanthin by LiAlH₄-reduction is described, confirming the structure proposed by Strain. 5. The identity of heteroxanthin with the trollein—like pigment described for Euglena is discussed.     

Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO₂ after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.     

Thiamin uptake in Euglena gracilis

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.     

Activation of Coupling Factor 1 from Euglena gracilis Chloroplasts : Conditions for Optimal Activation and Their Possible Physiological Significance

The recently described method for the activation of the Ca²⁺-ATPase of coupling factor 1 from chloroplasts (CF₁) of Euglena gracilis by low pH occurs optimally in high concentrations of NaCl, and is unaffected by the acid used to lower the pH to 4.5. Activation is inhibited by light, and this effect can be reversed by the presence of NADP⁺, ADP + inorganic phosphate, or an uncoupler. There appears to be no difference between the activities in the soluble and the particulate phases, and they seem to represent the same enzyme. The response of the activation process to light and to effectors of electron transport and phosphorylation indicates a possible physiological role for the acid activation of Euglena CF₁.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

Physiological adaptation of Euglena gracilis to uncouplers and inhibitors of oxidative phosphorylation

Cells of Euglena gracilis which have adapted to growth in the presence of 2,4-dinitrophenol are at the same time resistant to the inhibitory effect on growth of carbonylcyanide m-chlorophenylhydrazone, 1,7-hexafluoro-2,6-dihydroxy-2,6-bis(trifluoromethyl)-4 hep-tanone (“1799”), oligomycin, and tri-n-butylchlorotin. Cells adapted to any of the latter are at the same time resistant to dinitrophenol.The adaptation is a response to the primary effects of the uncouplers, i.e., high levels of ADP, and not to the uncouplers per se, since arsenate induced uncoupler resistance while nonuncoupling substituted phenols did not.No evidence for any protein in the cells which can bind uncouplers tightly could be found, and no modification of reactions of isolated mitochondria could be detected. Substrate utilization by adapted cells was only slightly less efficient than in normal cells.The inhibitors were accumulated by and not excluded from the cells. Thus, the cross-resistance implies either the formation of a modified mitochondrial membrane, impermeable to these hydrophobic effectors, or an alternative or greatly modified pathway for ATP synthesis, insensitive to these effectors.     

Ribosomal RNA cistrons in Euglena gracilis

Euglena gracilis chloroplasts contain about 12 fg DNA of average density 1.686 g cm^?3 and 1.7 pg RNA. The large (1.1 × 10⁶ mol. wt) and small (0.56 × 10⁶ mol. wt) ribosomal RNA components are coded for by separate cistrons, both of which band at a density of 1.696 g cm^?3 in a CsCl gradient. About 6% of the chloroplast DNA codes for rRNA indicating that there are 240 cistrons for rRNA in each chloroplast or about three to six cistrons per chloroplast genome. Similar studies with rRNA from cytoplasmic ribosomes indicate that the cistrons for cytoplasmic rRNA band at a density of 1.716 g cm^?3, denser than that of the main-band DNA, and that there are 1000 cistrons for cytoplasmic rRNA per cell. Fractionation of E. gracilis DNA on CsCl gradients and subsequent hybridization experiments, as well as melting curves of DNA-RNA hybrids, show that chloroplast rRNA does not anneal specifically with either the cistrons for cytoplasmic rRNA or any DNA in the dark-grown cell, in contrast to those results found in some higher plants.     

Guanine Metabolism in Carbon-Limited Euglena gracilis

SYNOPSIS. Guanine-8-³H is not a specific label for nucleic acids in Euglena gracilis (strain SM-L1, streptomycin-bleached), but is incorporated into all biochemical fractions. Autoradiographic results show that guanine is metabolized in, both the nucleus and the cytoplasm of these Eugelna , but nuclear metabolism of guanine occurs only at a very low level in carbon-limited cells. Further, carbon-limited Euglena incorporate less total guanine than do control Euglena in a comparable time period. However, the majority of label incorporated into the nucleic acid fraction of the carbon-limited cells is incorporated into RNA.     

Dark-induced chloroplast dedifferentiation in Euglena gracilis

Transfer of light-grown autotrophic Euglena gracilis cells to darkness and carbon (glucose) containing heterotrophic media causes structural and functional decomposition of the photosynthetic apparatus. The process can be ascribed to a strict diluting-out mechanism of stroma constituents among the progeny, as shown for ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), and aminoacyl-tRNA synthetases (Aa-RS; especially Leu-RS, EC 6.1.1.4) activities. The diluting-out effect of thylakoid membranes and chlorophyll seems to be superimposed by additional degradations, beginning soon after the transfer of cells to darkness. Cultivation of cells in darkness in 0.03 M KCl or without utilizable organic carbon (resting media) preserves chloroplast structure and function over a long period, indicating negligible turnover in these cells. Thus, under both growing and resting conditions, darkness induces the arrest of synthesis of plastid constituents. Experiments with the inhibitors cycloheximide, chloramphenicol, and nalidixic acid demonstrate that chloroplast dedifferentiation does not require organelle gene expression, but it is more strictly dependent on biosynthetic events in the nucleo-cytoplasmic compartment than the reverse process, light-induced chloroplast formation. Since cycloheximide at low concentrations in growth medium causes a marked suppression of precursor uptake or re-utilization similar to that in cells of resting media, intracellular precursor deficiency is suggested to control the observed blockade in cytoplasmic synthesis of plastid proteins. On the other hand, darkness might signalize the stop of gene expression in the organelles.Abbreviations Aa aminoacid - CH cycloheximide - CM chloramphenicol - Leu-RS leucyl-tRNA synthetase - RuBP ribulose-1,5-bisphosphate - TCA trichloroacetic acid     

Biosynthesis of polyamines in Euglena gracilis

In Euglena gracilis Z the biosynthesis of spermidine and spermine closely resembles the pathways occurring in mammalian tissues and in most microorganisms. l-Ornithine and not l-arginine, as is the case in most plants, is the main precursor of putrescine, and S-adenosylmethionine donates the propylamino moiety for the biosynthesis of spermidine and spermine. Cell-free extracts of Euglena synthesized sym-norspermidine and sym-norspermine from 1,3-diaminopropane and labelled S-adenosylmenthionine. The synthases for the biosynthesis of these two polyamines have a pH optimum of 7.6, like that of spermidine and spermine synthases. Ion exchange chromatography showed two peaks corresponding to the retention times of 2,4-diaminobutyric acid and 1,3-diaminopropane, lower homologues of ornithine and putrescine, respectively. Experiments with dl-2,4-diaminobutyric acid-[4-¹⁴C] did not result in significant incorporation of the label into 1,3-diaminopropane.