NADH-Dependent Glutamat Synthase in Euglena gracilis z

Novel antioxidative phenylpropanoid-substituted tocopherol derivatives, prunusols A and B, were isolated from the leaf wax of Prunus grayana Maxim., and their structures were fully characterized by spectroscopic and synthetic methods. Prunusols A and B were found to be the conjugates of γ-tocopherol and p-coumaric acid, which are diastereoisomers of each other. They showed almost the same antioxidative activity as α-tocopherol in a water/alcohol system measured by thiocyanate and TBA methods.     

Preparation of Physiologically Intact Mitochondria from Euglena gracilis z

A fluorescent reagent, N-(9-acridinyl)maleimide (NAM), was used for the determination of thiols in biological samples by high performance liquid chromatography. NAM-labeled glutathione (GSH), homocysteine, coenzyme A (CoA) and cysteine (CySH) were separated on a reversed-phase partition (octadecylsililated silica gel) column with the elution conditions of 0.06 m borate buffer pH 8.8: methanol (13: 1) at a flow rate of 0.6 ml/min within 15 min. In the absence of CoA in the sample, the elution conditions of 0.1 m borate buffer pH 8.8: methanol (15: 1) at a flow rate of 0.8 ml/min was used for the separations. Calibration curves were held up to 2.5 pmol for GSH and 11 pmol for CySH. About 0.17 µl of rat blood and 0.03 mg of rat liver equivalent to 0.1 nmol of GSH were determined. The sensitivity was 100 times higher than that obtained with an automatic amino acid analyzer.     

Isolation of Mutants of Euglena gracilis With Impaired Photosynthesis

Four mutant strains of Euglena gracilis have been isolated after treatment of wild type cells with ultraviolet light or the chemical mutagen nitrosoguanidine. None of the mutants is capable of autotrophic growth or photosynthetic carbon dioxide fixation.The mutant strains contain normal amounts of the enzymes of the reductive pentose phosphate cycle and are qualitatively similar to the wild type in pigment composition, but are unable to carry out the Hill reaction (light induced reduction of 2,6-dichlorophenol indophenol). Isolated mutant plastids cannot photoreduce NADP with water as the electron donor but can carry out this reaction when the electron donating system is ascorbate and 2,6-dichlorophenol indophenol. Whole cells of the mutants show the light induced oxidation of cytochrome f by light reaction I but are unable to bring about cytochrome f reduction by light reaction II. The mutants appear to be blocked at or near light reaction II in the photosynthetic electron transport chain.The mutants may represent alterations of the chloroplast genome since the mutation isolation was carried out under conditions where chloroplast viability was severely impaired, but cell viability was unaffected.     

Mechanism of metabolic regulation in photoassimilation of propionate in Euglena gracilis z

Euglena gracilis showed a typical photoassimilation of propionate when cultured on propionate as a sole carbon source. While the acid is metabolized by the methylmalonyl-coenzyme A (CoA) pathway under illumination, supporting growth of Euglena (K. Hosotani, A. Yokota, Y. Nakano, and S. Kitaoka, 1980, Agr. Biol. Chem.44, 1097–1103), it does not allow the protozoon to grow in the dark although it was actively taken up and metabolized. Kinetics of incorporation of radioactivity of labeled propionate, trapping effect of exogenous lactate in the incorporation of labeled propionate and radiorespirometric pattern revealed that propionate was metabolized by the lactate pathway in Euglena in the dark. Enzymes involved in the lactate pathway were located in mitochondria. The reason why Euglena can not grow on propionate in the dark is explained by the failure of producing C₄ dicarboxylic acids essential for biosynthesis of amino acids and sugars, like the mitochondrial oxidation of fatty acids in higher animals. The Euglena cells cultured in the dark contained enzymes of both methylmalonyl-CoA and lactate pathways, but lack of photosynthetically generated ATP has been suggested to force Euglena to select the less-ATP-requiring but futile pathway.     

Characterization and physiological function of glutathione reductase in Euglena gracilis z.

The purified glutathione reductase was homogeneous on polyacrylamide-gel electrophoresis. It had an Mr of 79,000 and consisted of two subunits with a Mr of 40,000. The activity was maximum at pH 8.2 and 52 degrees C. It was specific for NADPH but not for NADH as the electron donor; the reverse reaction was not observed. The Km values for NADPH and GSSG were 14 and 55 microM respectively. The enzyme activity was markedly inhibited by thiol inhibitors and metal ions such as Hg2+, Cu2+ and Zn2+. Euglena cells contained total glutathione at millimolar concentration. GSH constituted more than 80% of total glutathione in Euglena under various growth conditions. Glutathione reductase was located solely in cytosol, as were L-ascorbate peroxidase and dehydroascorbate reductase, which constitute the oxidation-reduction cycle of L-ascorbate [Shigeoka et al. (1980) Biochem. J. 186, 377-380]. These results indicate that glutathione reductase functions to maintain glutathione in the reduced form and to accelerate the oxidation-reduction of L-ascorbate, which scavenges peroxides generated in Euglena cells.     

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.     

Purification and Characterization of Serine Hydroxymethyltransferase from Mitochondria of Euglena gracilis z

Mitochondrial serine hydroxymethyltransferase, l-serine: tetrahydrofolate 5,10-hydroxymethyl-transferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with l-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88,000 by gel filtration through Sephadex G-200, and 44,000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5′-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had l-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity.     

Isolation, purification, and characterization of the pellicle of Euglena gracilis z

The pellicle was isolated from the cell homogenate obtained on sonication of Euglena gracilis z grown aerobically under illumination and purified by a combination of differential and sucrose density gradient centrifugations. The purity and homogeneity of the pellicle fragments were determined by an electron microscopic method and biochemical analysis of the components. The protein, lipid, and sugar contents of the purified pellicle were 68.7, 17.9, and 13.5%, respectively. The equilibrium density of pellicle fragments was 1.21 g/cm3. SDS-polyacrylamide gel electrophoresis revealed that the pellicle contained 50 mol% of nonpolar amino acids. The constituents of the lipid and sugar were very different from those of the cell membrane of other organisms.     

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.     

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)     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.     

Culture pH, CO2 tension, and cell division in Euglena gracilis Z.

Growth characteristics of Euglena gracilis Z as functions of culture pH, CO2 tension, temperature, and lighting regime were investigated. The results are consistent with the possibility that cell division is preceded by a lowered intracellular pH. Also consistent with this possibility is the finding that division rhythmicity can be induced by periodic changes in CO2 tension. It is suggested that the rhythmicity is induced by changes in intracellular pH produced by carbonic acid.     

Different metabolic fate of two carbons of glycolate in its conversion to serine in Euglena gracilis z

In our preceding work (A. Yokota, Y. Nakano, and S. Kitaoka, 1978, Agric. Biol. Chem. 42, 121-129), extensive decarboxylation of glycolate carboxyl carbon during its metabolism in Euglena gracilis suggested occurrence of a metabolic pathway of glycolate different from that of higher C3 plants. In the present report, we establish the Euglena glycolate pathway from characteristics of the decarboxylation of the carboxyl carbon and from the metabolic fate of hydroxymethyl carbon of glycolate. The ratio of the decarboxylation of the carboxyl carbon of glycolate to the total metabolized carbon increased with increasing metabolic rate in an asymptotic fashion. Thus, the ratio was 20% at the metabolic rate of 0.05 nmol of glycolate/10(6) cells/min, but it was over 60% at the rate of more than 0.35 nmol/10(6) cells/min after 2 min of incubation. Metabolic products were also changed depending on the rate of metabolism of glycolate; glycine was the main product at the low rate of glycolate metabolism and the contribution of glycine was reversed by the increased contribution of evolved CO2 at the high rates. At the metabolic rate of 1.5 nmol of glycolate/10(6) cells/min, the rate of the decarboxylation was 1.0 nmol of CO2/10(6) cells/min, which could not be explained by the extremely low activity of glycine synthase in Euglena. Experiments with [2-14C]glycolate showed that exogenously added formate and methionine caused accumulation of radioactive formate. Based on these results, we have proposed that the glycolate metabolism of E. gracilis consists of glycine and formate pathways and that the relative contribution of both pathways to the glycolate metabolism depends on the metabolic rate of glycolate.     

Peroxidative Activity in Euglena gracilis

Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3-amino-1,2,4-triazole.     

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.     

Photosynthetic Activity during the Division Cycle in Synchronized Euglena gracilis

Axenic populations of the photosynthetic protozoan Euglena gracilis, grown with autotrophic nutrition, were synchronized with respect to cell division by culture on an alternating light-dark cycle. No cell divisions occurred in the light periods; approximately 100% of the cells divided in the dark periods. In such cultures, the synthesis of photosynthetic pigments and accumulation of polysaccharide were confined to the light periods. The capacity for photosynthesis, however, increased continuously over the entire light-dark cycle, and is thus not directly correlated with pigment content. A correlation was seen between photosynthetic capacity and protein content, suggesting that enzymatic mechanisms of the photosynthetic apparatus might be the limiting factor. Estimates of total photosynthetic activity indicate that about 5 x 10(-6) calories are required for the synthesis of a new cell.