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G A Cross

Publications and source records attributed to G A Cross.

17 recordsLinked to original sources
PubMed1992

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.

Animals↗
PubMed1979

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.

Amino Acids↗
PubMed1977

The synthesis of a variant-specific antigen by Trypanosoma brucei in vitro.

A variant-specific surface antigen from a cloned population of Trypanosoma brucei S42 has been isolated and partially characterized. [35S]L-methionine was found to be incorporated into this material by cells incubated in vitro in a chemically defined medium. Incorporation of [35S]L-methionine was inhibited by cycloheximide and puromycin at concentrations which are known to specifically inhibit protein synthesis in other systems. The rate of synthesis of the variant-specific antigen in vitro has been estimated to be about 8% of the rate in vivo. Newly synthesized [35S]L-methionine-labelled variant-specific antigen was incorporated into the surface coat.

Animals↗
PubMed1977

Threonine catabolism in Trypanosoma brucei.

L-Threonine is catabolized by Trypanosoma brucei to give equimolar quantities of glycine and acetate. The pathway, which involves the two enzymes L-threonine dehydrogenase (EC 1.1.1.103) and aminoacetone synthase (acetyl-CoA:glycine C-acetyltransferase, EC 2.3.1.29) and subsequent hydrolysis of the acetyl-CoA, is most active in cultured trypanosomes but is also present in bloodstream forms. L-Threonine dehydrogenase from both culture and bloodstream forms of trypanosomes has an apparent molecular weight of between 28 000 and 38 000, and is sensitive to a wide range of sulphydryl reagents.

Acetone↗
PubMed1977

Isolation of mitochondria and mitochondrial RNA from Crithidia fasciculata.

Two methods were used to isolate mitochondria from Crithidia fasciculata. In the first method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the second, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined with the electron microscope and were also shown to possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA. Analysis by polyacrylamide gel electrophoresis showed two RNA components of molecular weights of 0-47 X 10(6) and 0-22 X 10(6), in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14-6S, 11-4S, 10-1S and 9-9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H]uridine into the presumed mitochondrial RNA.

Cell Fractionation↗
PubMed1977

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei.

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).

Animals↗
PubMed1976

Lability of RNA from the large cytoplasmic ribosomal subunit of the protozoon Crithidia oncopelti.

Cytoplasmic ribosomalRNA extracted from Crithidia oncopelti and analysed by gel electrophoresis at 4 degrees C consisted of two components, with molecular weights (relative to E. coli rRNA) of 1-30 X 10(6) and 0-83 X 10(6) daltons, present in equimolar amounts. On heating briefly at 51 degrees C followed by rapid cooling, the 1-30 X 10(6) RNA completely dissociated into two components of molecular weights 0-70 X 10(6) and 0-56 X 10(6) (present in equimolar amounts). Fifty per cent dissociation of the molecule occurred at 28 degrees C. That the integrity of the RNA molecule at low temperatures is maintained by its secondary structure was confirmed by electrophoresis under denaturing conditions (98%, v/v, formamide). To account for these phenomena, latent cleavage of the molecule in vivo is proposed.

Animals↗
PubMed1975

Purification and properties of nucleic acids from an unusual cytoplasmic organelle in the flagellate protozoan Crithidia oncopelti.

A simple, rapid method for preparing bipolar bodies from sonicated (rithidia oncopelti cells, with a yield of 2-5%, is described. Apart from 2-4% contamination with unbroken cells the fraction was considered pure with respect to contamination by other nucleic acid-containing organelles, as judged by light and electron microscopy. A light satellite DNA, f bouyant density 1.695 g/ml in neutral CsCl, and derived from the bipolar body, had a Tm of 81.6 degrees C in0.15 M NaCl/0.015 M sodium citrate (pH 7.0) and a kinetic complexity of 2.7 with 109. The bipolar body fraction also contained ribonucleoprotein particles with and s20,w of 67 S, in contrast to cytoplasmic ribosomes (87 S). Bipolar body ribosomes contained rRNA components which migraged coincidentally with Escherichia coli rRNA (molecular weights 1.07 with 10-6 and 0.56 with 10-6) on polyacrylamide gel electrophoresis. Cytoplasmic ribosomes contained rRNAs of molecular weights 1.30 with 10-6 and 0.83 with 10-6. Bipolar body rRNA accounted for up to 10% of the rRNA extracted from cells. The properties of these bipolar body nucleic acids provide good evidence for the bacterial nature of this subcellular component.

Animals↗
PubMed1975

Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate.

The amino acid compositions of several culture media have been analysed and compared. The utilization and excretion of amino acids and other metabolites have been followed during growth of Trypanosoma brucei S42 in a defined medium. All of the added L-threonine was metabolized by the cells, even when it was present at elevated concentrations. Glucose was consumed throughout the growth cycle: glutamine was consumed more rapidly than glutamic acid, which was itself used at about the same rate as proline. Threonine was cleaved to form glycine and acetate, both of which accumulated in the medium. Alanine and succinate were excreted together with a small amount of pyruvate, but these three products accounted for less than half of the glucose used. CO2 production from glucose was not measured, but insignificant amounts of CO2 were produced from threonine. Tetraethylthiuram disulphide blocked the cleavage of threonine and was a potent inhibitor of trypanosome growth.

Acetates↗
PubMed1975

Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei.

Soluble glycoproteins have been purified from a series of clones of Trypanosoma brucei 427. Each clone yielded a characteristic predominant glycoprotein which induced clone-specific immunity to trypanosome infection in mice. These glycoproteins were shown by specific labelling and enzyme digestion of cells to be the major components of the trypanosome surface coat. Each glycoprotein consisted of a single polypeptide chain having an apparent molecular weight of 65 000 (as measured by SDS-polyacrylamide gel electrophoresis) and containing around 600 amino acid and 20 monosaccharide residues. Preliminary structural studies indicated large changes in amino acid sequence dispersed over a considerable length of the polypeptide chain. Proteolytic activity was demonstrated in semi-purified trypanosome extracts, providing one reason for the heterogeneity sometimes observed in surface glycoprotein antigen preparations.

Animals↗