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.     

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.     

Purification and some properties of mitochondrial glutamate:glyoxylate aminotransferase and mechanism of its involvement in glycolate pathway in Euglena gracilis z

Euglena contains glutamate:glyoxylate aminotransferase (GGT) both in mitochondria and in cytosol. Both isoforms were separated from each other by DEAE-cellulose chromatography. The mitochondrial enzyme had an apparent Km of 1.9 mM for glutamate and the cytosolic enzyme 52.6 mM. Mitochondrial GGT was further purified by ammonium sulfate fractionation, isoelectric focusing, and gel chromatography. It had a molecular weight of 141,000 and an isoelectric point of pH 4.88; the optimum pH was 8.5. Its apparent Km values for glutamate and for glyoxylate were 2.0 and 0.25 mM, respectively. In addition to glutamate, mitochondrial GGT used 5-hydroxytryptophan, tryptophan, and cysteine as amino donors in the transamination to glyoxylate. Alanine did not support the activity. The relative activity of the enzyme for amino acceptors on the transamination from glutamate was 4-hydroxyphenylpyruvate greater than phenylpyruvate greater than glyoxylate greater than hydroxypyruvate. Pyruvate and 2-oxoglutarate were not used in the reaction. Evidence that GGT functions mainly in the irreversible transamination between glutamate and glyoxylate is presented. The functional significance of GGT in the glycolate pathway of Euglena is also discussed.     

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.     

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.     

Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis

Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.     

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.     

Evaluation of two biosynthetic pathways to delta-aminolevulinic acid in Euglena gracilis.

delta-Aminolevulinic acid (ALA), which is an intermediate in the biosynthesis of chlorophyll a, can be biosynthesized via the C5 pathway and the Shemin pathway in Euglena gracilis. Analysis of the (13)C-NMR spectrum of (13)C-labeled methyl pheophorbide a, derived from 13C-labeled chlorophyll a biosynthesized from d-[1-(13)C]glucose by E. gracilis, provided evidence suggesting that ALA incorporated in the (13)C-labeled chlorophyll a was synthesized via both the C5 pathway and the Shemin pathway in a ratio of between 1.5 and 1.7 to one. The methoxyl carbon of the methoxycarbonyl group at C-132 of chlorophyll a was labeled with (13)C. The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3(1), C-P4, C-P6, C-P7(1), C-P8, C-P10, C-P11(1), C-P12, C-P14, C-P15(1) and C-P16.     

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.     

Periodic, metabolic and structural phenomena in a protist, Euglena gracilis]

Sychronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and ther periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous--this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G1, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G1 phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G1 phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.     

Comparison of two fatty acid synthetases from Euglena gracilis variety bacillaris

Two enzyme systems from Euglena gracilis var. bacillaris which catalyze the de novo biosynthesis of fatty acids have been compared. One is a multienzyme complex of high molecular weight which is independent of ACP for activity in vitro, and the other is an ACP-dependent system of discrete enzymes (M. L. Ernst-Fonberg, (1973) Biochemistry12, 2449–2455). The latter activity is present in small amounts in etiolated cells and increases upon exposure of dark-grown cells to light, while multienzyme complex fatty acid synthetase activity decreases by about one-half after 24 hr of exposure to light. Results from the greening of dark-grown cells in the presence of cycloheximide, chloramphenicol, or spectinomycin suggests that the chloroplast ribosomes are involved in the appearance of the ACP-dependent activity; alternatively, the cytoplasmic ribosomes appear to be the site of biosynthesis of the multienzyme complex fatty acid synthetase (or a protein responsible for its activation). The fatty acid synthetase activities from several chloroplast mutants were measured. The ACP-dependent activity was reduced or not present depending on the degree of impairment of chloroplast development, while the multienzyme complex activity in all instances continued to respond to light or darkness.Antibodies against the purified multienzyme complex extensively inhibited its activity whereas the activity of the ACP-dependent system was consistently stimulated. The two enzyme systems are immunologically cross reactive but not identical.     

Separation and properties of the NAD-linked and NADP-linked isozymes of succinic semialdehyde dehydrogenase in Euglena gracilis z.

Euglena gracilis z contained two succinic semialdehyde dehydrogenases (EC 1.2.1.16), one requiring NAD and the other NADP, and these isozymes were separated from each other and partially purified. The NAD-linked isozyme was relatively stable on storage at 5 degrees C whereas the NADP-linked one was extremely unstable unless 30% glycerol or ethyleneglycol was added. The optimum pH was 8.7 and optimum temperature 35-45 degrees C for both isozymes. They were inhibited by Zn2+ and activated, particularly the NAD-linked enzyme, by K+. Sulfhydryl reagents activated both isozymes. The Km values for succinic semialdehyde were 1.66 - 10(-4) M with the NAD-linked isozyme and 1.06 - 10(-3) M with the NADP-linked one. The NADP-linked isozyme was induced by glutamate while the NAD-linked one was not. Probable roles of these isozymes in the physiology of Euglena gracilis are discussed.     

Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts

Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5′-deoxyadenosylcobalamin, reached about 1 femtomole/10⁶ cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5′-deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5′-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes.     

Compartmentation of uracil in Euglena gracilis.

Compartmentation of uracil in the flagellate Euglena gracilis was studied by tracer-kinetic experiments. Lag times in the equilibration of exogenously given and intracellularly present uracil before linear labeling of catabolic and anabolic products was determined to estimate the size of its metabolically active pool. This pool operates in the incorporation and degradation of uracil. There were the same lag times in forming both final products when measured in parallel and when measured after preloading with pyrimidines, in different cell strains, and under various environmental conditions. The amount of the metabolically active uracil pool, estimated as 11 pmol/10(7) heterotrophically growing cells, decreased to almost zero during light-induced RNA synthesis and could be changed by preloading with uracil or thymine. Besides this metabolic pool, cells may contain large amounts of uracil in a membrane-enclosed storage compartment (up to 12 nmol/10(7) cells). This is metabolically inert, but may be mobilized by nitrogen-carbon starvation. The role of uracil compartmentation in this metabolically flexible organism is discussed.     

Quantitative Measurement of Paramylum in Euglena gracilis

SYNOPSIS. A technic is described for the quantitative assay of paramylum content of euglenoid flagellates. The method relies on the alkaline solubility of paramylum followed by treatment with the anthrone reagent. The intensity of the color developed by paramylum is about 14% greater than that developed by an equivalent amount of glucose. The method is sensitive down to about 10 μg.     

Maturation of chloroplast rRNA in Euglena gracilis

RNA and protein synthesis in the myocardium were stimulated after a short preincubation period with calcium. This elevation of macromolecular synthesis persisted in the absence of the ion for at least four hours. It appears that the uptake and/or the concentration of intracellular calcium induced a persistent and optimum enhancement of RNA and protein synthesis.