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Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight.

ABSTRACT: Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an 'activated'-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5'-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 X 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

SUBMITTER: McLennan AG

PROVIDER: S-EPMC1172351 | biostudies-other | 1975 Nov

REPOSITORIES: biostudies-other

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Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight.

Project description:The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme.

| S-EPMC1172352 | biostudies-other

Purification, properties and amino acid sequence of atypical cytochrome c from two protozoa, Euglena gracilis and Crithidia oncopelti.

Project description:A basic cytochrome was isolated from the phytomastigophorean protozoan Euglena gracilis and a similar protein from the zoomastigophorean protozoan Crithidia oncopelti. In both cases chromatography on CM-cellulose in first the reduced and then the oxidized form proved to be an efficient means of purification. The two cytochromes can be classed in the cytochrome c family but they have certain atypical features. The alpha peak of the absorption spectrum is shifted towards the red and is asymmetrical. The pyridine ferrohaemochrome has an alpha-peak maximum intermediate between that of c-type cytochromes and proteins containing protohaem IX. The test for free vinyl groups was positive. The amino acid sequences of the two cytochromes were determined. Attention is drawn in the text to those parts of the evidence that are less satisfactory. Both sequences are homologous with the family of cytochrome c, but are unusual in having only one cysteine residue so that the haem is attached through only one thioether bond. Detailed evidence for the amino acid dequences of the two proteins has been deposited as Supplementary Publication SUP 50042 (70 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.

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

Project description:Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.

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

Project description: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.

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Presence of two deoxyribonucleic acid polymerases in bull spermatozoa.

Project description:A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.

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The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Partial purification and characterization of four distinct activities.

Project description:Four extramitochondrial DNA polymerases from the marine photosynthetic diatom Cylindrotheca fusiformis were isolated and purified more than 1200-fold by chromatography on DNA-cellulose and DEAE-Sephadex. The enzymes were equally susceptible to inhibition by the thiol-blocking agents N-ethylmaleimide and p-chloromercuribenzoate, the zinc chelator o-phenathroline, and the nucleic acid interchelators ethidium bromide and acriflavin; they displayed similar pH optima, preferred activated DNA, and had strict dependence on high K+ for maximum activity. They were differentiated on the basis of their kinetic parameters, template-primer utilization and salt requirements. The four activities varied with growth stage of C. fusiformis. Activities of polymerases A and D doubled in exponential-phase cells as compared with those in stationary-phase cells, and the increase in polymerase B and chloroplast activity C was 20-40%. The relationship of the diatom polymerases to the complements in other organisms is discussed.

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Sequence evidence for the presence of two tetrapyrrole pathways in Euglena gracilis.

Project description:Genes encoding enzymes of the tetrapyrrole biosynthetic pathway were searched within Euglena gracilis EST databases and 454 genome reads and their 5' end regions were sequenced when not available. Phylogenetic analyses and protein localization predictions support the hypothesis concerning the presence of two separated tetrapyrrole pathways in E. gracilis. One of these pathways resembles the heme synthesis in primarily heterotrophic eukaryotes and was presumably present in the host cell prior to secondary endosymbiosis with a green alga. The second pathway is similar to the plastid-localized tetrapyrrole syntheses in plants and photosynthetic algae. It appears to be localized to the secondary plastid, presumably derived from an algal endosymbiont and probably serves only for the production of plastidial heme and chlorophyll. Thus, E. gracilis represents an evolutionary intermediate in a metabolic transformation of a primary heterotroph to a photoautotroph through secondary endosymbiosis. We propose here that the tetrapyrrole pathway serves as a highly informative marker for the evolution of plastids and plays a crucial role in the loss of plastids.

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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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The purification and amino acid sequence of cytochrome C-552 from Euglena gracilis.

Project description:Cytochrome c-552 from Euglena gracilis was purified and the amino acid sequence determined. The protein is a single peptide chain of 87 residues with the haem prosthetic group bound through two thioether linkages to two cysteine residues near the amino-terminal region. The amino acid sequence shows some similarities to mitochondrial cytochrome c and to two prokaryote c-type cytochromes. The sequence, taken with the known characteristics of cytochrome c-552, indicates that it is an f-type cytochrome. The possible functional and evolutionary significance of these features in common is discussed. Detailed evidence for the amino acid sequence of Euglena cytochrome f has been deposited as Supplementary Publication SUP 50027 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

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