Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

Steiger, R.; Opperdoes, Frederik; Bontemps, José

doi:10.1111/j.1432-1033.1980.tb04486.x

Cited by 133 publications

(70 citation statements)

References 49 publications

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“…This leads to an inhibition of the shuttle, which, in turn, would lead to a decrease in the labelling rate of the cytosol. Thirdly, about 50 of the total glyceraldehyde-phosphate dehydrogenase content of the cell is found in the cytosol [4,5,22] and we routinely find slightly higher percentages of glucose-phosphate and triosephosphate isomerase and 3-phosphoglycerate kinase in the soluble fractions than of the other enzymes. This would allow labelling of cell-sap glycolytic intermediates via the dihydroxyacetone phosphatelglycerol-3-P shuttle between glycosome and cell sap.…”

Section: Discussionmentioning

confidence: 94%

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Opperdoes²,

1981

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In cell -fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosomu brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360 -3641. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-'4C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s and represents 20-30 of total cellular metabolites. Radioactively labelled pyruvate is already produced after I5 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70-800/, of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol.Incorporation of radioactivity into the total glycerol 3-phosphate pool is more rapid than for the other metabolites studied. This indicates rapid mixing of the glycosomal and cell-sap pools of glycerol 3-phosphate, as required for the extra-glycosomal oxidation of glycerol 3-phosphate by the mitochondria1 oxidase.In the presence of 1 mM salicylhydroxamic acid, which mimics anaerobiosis, the trypanosome produces equimolar amounts of pyruvate and glycerol. Labelling of glycerol and glycerol 3-phosphate proceeds at identical rates but at all times the specific activity of glycerol is less than that of glycerol 3-phosphate. This is compatible with our earlier proposal that glycerol is made from glycerol 3-phosphate by glycerol kinase.We conclude that glycolysis in trypanosomes takes place in the glycosome and that the membrane of this organelle is poorly permeable to most glycolytic intermediates.African trypanosomes, like Trypunosoma brucei, are unicellular flagellate protozoa, that are well adapted to life in the mammalian bloodstream. For their energy needs these organisms rely entirely on glycolysis, which proceeds at an extremely high rate and leads to pyruvate excretion as the only end-product of glycolysis under aerobic conditions [l 1.The NADH formed in glycolysis is reoxidized by means of a mitochondria1 glycerol-3-phosphate oxidase and molecular oxygen. When this oxidase is completely inhibited by 1 mM salicylhydroxamic acid, the trypanosome manages to survive by converting glucose into equimolar amounts of glycerol and pyruvate with concomitant synthesis of ATP.A unique aspect of the glycolytic pathway in T. brucei is that part of it is located inside a microbody-like organelle called glycosome. We have previously shown [2 -51 that nine enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate, are almost exclusively contained in the glycosome. All glycosomal enzymes exhibit high latency in...

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Section: Discussionmentioning

confidence: 94%

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Opperdoes²,

1981

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“…Morphologically it is indistinguishable between the procyclic insect and mammalian bloodstream stages. It too is defined by a LAMP-like membrane glycoprotein called p67 [2,3], acidic hydrolases such as cathepsin-L and -B orthologues [4][5][6][7], and a recent survey of the genome data base revealed a full complement of V-ATPase subunit orthologues [8]. It is well established that endocytosis is greatly upregulated in the bloodstream stage relative to procyclic forms [9][10][11], and this is mirrored by upregulation of lysosomal hydrolytic activities [6,7,9,12].…”

Section: Introductionmentioning

confidence: 99%

A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes

McCann

Schwartz

Bangs

2008

Molecular and Biochemical Parasitology

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The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH 4 Cl) then allows precise titration of steady state lysosomal pH (4.84 ± 0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa. Keywordstrypanosome; lysosome; flow cytometry; pHThe eukaryotic lysosome has been traditionally defined as a discrete terminal degradative organelle of the endocytic pathway with a high density (r = 1.10 g/ml) and a low internal pH (≤ 5.0), [1]. It contains a unique complement of structural membrane glycoprotein markers (lysosome-associated membrane proteins; LAMPs), acid hydrolases (lipases, proteases, phosphatases and glycosidases), and a membrane proton pump (V-ATPase) that is responsible for lumenal acidification. In African trypanosomes the lysosome is a single discrete organelle located at a perinuclear position in the posterior end of the cell. Morphologically it is indistinguishable between the procyclic insect and mammalian bloodstream stages. It too is defined by a LAMP-like membrane glycoprotein called p67 [2,3], acidic hydrolases such as cathepsin-L and -B orthologues [4][5][6][7], and a recent survey of the genome data base revealed a full complement of V-ATPase subunit orthologues [8]. It is well established that endocytosis is greatly upregulated in the bloodstream stage relative to procyclic forms [9][10][11], and this is mirrored by upregulation of lysosomal hydrolytic activities [6,7,9,12]. This difference at least in part reflects issues of nutrient acquisition for each life cycle stage. Procyclic cells can rely on the hydrolytic environment of the tsetse midgut to provide solutes for transport, while the bloodstream stage must aggressively take up and digest host serum macromolecules. A secondary consideration is immune evasion; potentially lytic or opsonic immune complexes # Corresponding Author: J.D. Bangs, email: jdbangs@wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the...

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“…5 (1) is the glycosomal GAPDH from T. brucei. For the calculation of percentage homology the areas containing insertions or deletions have not been taken into account (la) T. brucei cytosolic (2) E. coli (3) B. stearothermophihs (4) T. aquaticus (5) Yeast (6) Drosophila melanogaster (7) Lobster (8) Chicken (9) pig (10) Rat (11) brucei cytosolic GAPDH, therefore, must await the elucidation of its complete sequence. The large difference in sequence of the two T. brucei GAPDH isoenzymes is at complete variance with what we have previously found in the case of the glycosomal and cytosolic isoenzymes of phosphoglycerate kinase.…”

Section: Y D S V H G K F K H S V S T T K S K P S V a K D D T L V~' N mentioning

confidence: 99%

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Misset

Beeumen²,

Lambeir

et al. 1987

European Journal of Biochemistry

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Trypanosoma bruceicontains two glyceraldehyde-phosphate (GAPDH; EC 1.2.1.12) isoenzymes; one is located in glycosomes and represents 80% of the total activity, whereas the other is present in the cytosol. The purification of the cytosolic GAPDH, which is identical in both bloodstream-form and insect-stage trypanosomes, is described, and the enzyme compared with its glycosomal counterpart. Cytosolic GAPDH is specific for NAD. It is a tetrameric enzyme with subunits of 33.5 kDa, 5 kDa smaller than those of the glycosomal GAPDH. The native enzyme has a pl of 7.9, which is 1.5 pH units less basic than the glycosobal enzyme. Both enzymes display maximal activity at pH 8 but the cytosolic enzyme has a much broader activity profile especially towards lower pH values.Sequence comparison of the first 85 amino acids reveals that the N-terminal parts of both isoenzymes differ by 52%. The N terminus of the cytosolic isoenzyme resembles the corresponding N termini of ten other known GAPDH sequences in that they all lack three amino-acid insertions, which so far only have been found in the glycosomal isoenzyme of T. brucei. This observation explains in part the great difference in subunit size between the two T. brucei isoenzymes and suggests that at least one of these insertions is responsible for import of the glycosomal isoenzyme into the organelle.In Trypanosoma brucei, glyceraldehyde-phosphate dehydrogenase (GAPDH) has been found in two separate cellular compartments: the cytosol and the glycosomes [l]. However, thus far no direct proof has been brought forward for the existence of two separate isoenzymes in this organism.Recently two tandemly linked identical genes coding for GAPDH have been identified in the genome of T. brucei [2]. From sequence data it was inferred that both genes code for the glycosomal enzyme; subunit molecular mass, isoelectric point 131, amino-acid composition and the N-terminal sequence of the isolated glycosomal protein [2] were all in excellent agreement with the properties predicted from the amino acid sequence as deduced from the gene. A gene coding for a possible cytosolic isoenzyme has not yet been identified.In this paper we now present evidence for the existence of a separate cytosolic isoenzyme of GAPDH in cultured procyclic (insect-stage) as well as in bloodstream-form trypanosomes. We describe its purification, characterization and N-terminal amino acid sequence and compare the properties of both the glycosomal and the cytosolic counterparts.

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Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

Cited by 133 publications

References 49 publications

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

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