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Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.

ABSTRACT: In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

SUBMITTER: Watanabe F

PROVIDER: S-EPMC1148466 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis.

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Exploring the Glycans of Euglena gracilis.

Project description:Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.

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A Uniquely Complex Mitochondrial Proteome from Euglena gracilis.

Project description:Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

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Project description:Malate synthase (EC 4.1.3.2) from dark-grown Euglena gracilis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. The enzyme was released from acetate-grown cells by treatment with ultrasonic waves and purified from broken-cell suspensions by high-speed centrifugation and (NH4)2SO4 fractionation, followed by gel-filtration on Sepharose 6B. The final enzyme preparation was purified 190-fold compared with the crude extract. The mol.wt. of the enzyme was about 350000 as determined by gel filtration on Sepharose 6B. Treatment with sodium dodecyl sulphate and urea dissociated the enzyme into subunits of mol.wt. 175000. The pH optimum for the enzyme was 8.0 and the Km values for glyoxylate and acetyl-CoA were 50 and 80 micron respectively. Antibodies raised to the purified enzyme were shown to be monospecific by radiochemical immunoassay. Euglena anti-(malate synthase) tested on Ouchterlony double-diffusion gels gave a sharp precipitation band against acetate-grown Escherichia coli, but no immunological correspondence was observed with acetate-grown Chlorella fusca, Zea mays (maize) scutella or purified malate synthase from Ricinus communis.

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