Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K⁺ and Mg²⁺. Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca²⁺ are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca²⁺-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg²⁺.     

Purification and Immunological Properties of NAD+-linked Malate Dehydrogenase Isozymes from Euglena gracilis

We investigated the hypolipidemic effects of young persimmon fruit (YP) on apolipoprotein E-deficient C57BL/6.KOR-ApoE^shl mice. These mice exhibited higher plasma cholesterols, except for high-density lipoprotein (HDL), and lower plasma HDL cholesterol than C57BL/6.Cr mice that had the same genetic background as the C57BL/6.KOR-ApoE^shl mice. Male C57BL/6.KOR-ApoE^shl mice (n=5) were fed a diet supplemented with dry YP, Hachiya-kaki, at a concentration of 5% (w/w) for 10 weeks. YP treatment significantly lowered plasma chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterols, and triglyceride, and this response was accompanied by an elevation of fecal bile acid excretion. In the liver, sterol regulatory element binding protein-2 gene expression was significantly higher in mice fed YP, while the mRNA and protein levels of the LDL receptor did not change. These results indicate that acceleration of fecal bile acid excretion is a major mechanism of the hypolipidemic effect induced by YP in C57BL/6.KOR-ApoE^shl mice.     

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.     

Properties of Photosynthetic Mutants Isolated from Euglena gracilis

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.     

Some Properties of Carbamoyl Phosphate Synthase Partially Purified from the Larvae of Blowfly,Aldrichina grahami

Glutamine and ornithine were found to stabilize effectively carbamoyl phosphate synthase (CPSase) partially purified from the larvae of Aldrichina grahami reared aseptically.

Glutamine, ATP. and Mg ion were required for the enzyme reaction. A high concentration of ammonia could replace the requirement of glutamine; N-acetylglutamate could not enhance the reaction. The apparent Km for ammonium ion, however, was much higher than that for glutamine. The concentration of ATP required for half maximal velocity was 1.0×10^?2 m.

Various kinds of nucleotides of pyrimidines and purines inhibited the enzyme reaction. The reaction product in the assay system radioautographically coincided with citrulline.     

delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity

δ-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.

Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.

In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t_1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.

Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.

In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t_1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O₂ production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.

The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route.

     

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.     

Actin Gene Sequence from Euglena gracilis

ABSTRACT The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1.238 nucleotides, of which 1.131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.     

The alternative oxidase from Euglena gracilis

We still have a rudimentary understanding about the mechanism by which plant roots may stimulate soil microbial interactions. A biochemical model involving plant-derived biochemical fractions, such as exudates, has been used to explain this "rhizosphere effect" on bacteria. However, the variable response of other soil microbial groups, such as protozoa, to the rhizosphere suggests that other factors could be involved in shaping their communities. Thus, two experiments were designed to (a) obtain a better understanding of the mechanism by which ciliate species richness and abundance differ among plant species and (b) to determine whether this mechanism is maintained via stimulatory and/or inhibiting factors associated with particular plant species. Bacterial and chemical slurries were reciprocally exchanged between two plant species known to differ in terms of ciliate species richness and abundance (i.e., Canella winterana and plantation Tectona grandis ). The ANOVA showed that the bacteria plus nutrients, and the nutrients-only treatment have no significant effect on the overall ciliate species richness and abundance when compared to the control treatment. However, the use of only colpodean species to increase the taxonomic resolution of treatment effects showed that bacterial slurries have a significant effect on colpodean ciliate species richness. These results suggest that for particular rhizosphere ciliates, biological properties, such as bacterial diversity or abundance, may have a strong influence on their diversity and possibly abundance. These results are consistent with a model of soil bacteria-mediated mutualism between plants and protozoa.     

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s_{20, w} at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.