Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

Opperdoes, Frederik; Aarsen, P. N.; Meer, C. van der; Borst, Piet

doi:10.1016/0014-4894(76)90082-5

Cited by 65 publications

(20 citation statements)

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“…Essentially similar results were obtained in the presence of 1 mM salicylhydroxamic acid, an inhibitor of respiration [8,15], except that the first phase is faster (< 10 s). There is no detectable lag phase for pyruvate under these conditions …”

Section: Resultssupporting

confidence: 76%

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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: Resultssupporting

confidence: 76%

“…Trypanosoma brucei stock 427 was used in all experiments [8,9]. The parasites were grown in 300-g Wistar rats.…”

Section: Growth and Isolation Of Trypanosomesmentioning

confidence: 99%

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Opperdoes²,

1981

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“…Growth and Isolation of the Organism : Bloodstream forms of Trypanosoma brucei stock 427 were grown in 300-8 Wistar rats (17,18). Blood was collected from animals showing high parasitaemias (usually 3-4 d after infection) by cardiac puncture under ether anaesthesia .…”

Section: Methodsmentioning

confidence: 99%

Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.

Opperdoes¹,

Baudhuin²,

Coppens³

et al. 1984

The Journal of Cell Biology

197

107

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Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis . Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12-to 13-fold purified glycosomes with an overall yield of 31% . These glycosomes appeared to be highly pure and contained <1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 um with a standard deviation of 0.03 um . The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume . The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1 . The purified glycosomal fraction had a very low DNA content of 0.18 hg/mg protein . No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.

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“…Under aerobic conditions, glucose cannot be oxidized beyond pyruvate, which appears to be the exclusive end-product of glycolysis [3,4], whereas under anaerobic conditions equimolar amounts of pyruvate and glycerol are excreted [3,5]. This anaerobic dis-624 Glycolysis in Trypanosoma brucei mutation of glucose, which can be mimicked by incubating trypanosomes in the presence of 1 mM salicylhydroxamic acid [6,7] proceeds with net ATP synthesis [7], an observation which cannot readily be explained from the data available at present.We have recently shown that in T. brucei a number of glycolytic enzymes and glycerol kinase are located inside a microbody-like organelle instead of being in the cytosol, as is the situation in all other eukaryotes investigated so far [8-101. This organelle, which we have called 'glycosome' for obvious reasons [lo], catalyses the conversion of glucose and glycerol into 3-phosphoglycerate (1) and (2) :…”

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confidence: 99%

Glycolysis in Trypanosoma brucei

Visser

Opperdoes

1980

European Journal of Biochemistry

122

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The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei FEBS Lett. 80, 360-3641 constitute a separate pool of glycolytic intermediates within the cell was investigated.In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell-sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities or the latter enzymes were all latent between 70 and 92 %. Latency was abolished by addition of 0.1 % Triton X-100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co-factors of glycolysis.The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes : (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol-3-phosphate, however, increased fourfold. From the high levels of metabolite concentrations found and from comparison of the apparent mass-action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.Bloodstream forms of the African trypanosomes belonging to the brucei group, like Trypanosoma brucei, Trypanosoma rhodesiense and Trypanosoma gambiense, are the causative agents of nagana in domestic animals Abbreviations. Dihydroxyacetone-P, dihydroxyacetone phosphate ; glycerol-3-P, sn-glycerol-3-phosphate; glycerate-3-P, 3-phosphoglycerate; glucose-6-P, glucose-6-phosphate; fructose-6-P, fructose-6-phosphate; fructose-l,h-Pz, fructose-1,6-bisphosphate; glycerate-2-P, 2-phosphoglycerate; glyceraldehyde-3-P, D-glyceraldehyde-3-phosphate.Enzymes. Hexokinase (EC 2.7.1.1); phosphoglucose isomerase (EC 5.3.1.9); 6-phosphofructokinase (EC 2.7.1.11); fructose-bisphosphate aldolase (EC 4.1.2.13); triosephosphate isomerase (EC 5.3.1 .l): sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); glycerol kinase (EC 2.7.1.30); glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3); phosphoglyceromutase (EC 2.7.5.3); enolase (EC 4.2.1.11); pyruvate kinase (EC 2.7.1.40); alanine aminotransferase (EC 2.6.1.2). and live-stock and of the acute and chronic forms of sleeping sickness in man. For their energy needs, these organisms are entirely dependent on glycolysis which proceeds at an extremely high rate and differs in its...

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Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

Cited by 65 publications

References 10 publications

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.

Glycolysis in Trypanosoma brucei

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