Glycolysis in Trypanosoma brucei

Abstract

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...

Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

“…T. brucei was incubated in vitro under aerobic conditions in a glucose-containing mixture which allows the maintenance of a high intra-cellular ATP/ADP ratio [5]. Samples were taken 5 s to 10 min after adding the radioactive glucose and the radioactivity was determined in hexose monophosphates, fructose bisphosphate, phosphoglycerate and phosphoenolpyruvate, the intermediates most clearly separated on the anion-exchange column.…”

“…The parasites were separated from blood components by centrifugation at 600 x g for 30 min. The trypanosomes were collected from the interface and washed twice with glucose-containing phosphate-saline buffer and further purified according to Lanham [5]. The effluent was split into two parts.…”

“…The other part was andlysed for total phosphate by an automated phosphate determination [5, 131. The reagents for the phosphate determination were modified as follows: solution A: 0.36 M HzS04, 31 mM ammonium heptamolybdate and 1 ml Liqui-Nox/l ; solution B: 0.36 M HzS04, 68 mM L-ascorbic acid, 15 mM potassium antimony(I1I)tartrate and l ml Liqui-Nox/I. Peaks in the chromatogram were identified by co-chromatography of known standards and by enzymic determination [5]. Specific activities of metabolites were calculated in pooled peak fractions by enzymic analysis and by the measurement of radioactivity in the same sample.…”

“…The effluent was split into two parts. One part was used for the enzymic determination of metabolites as described previously [5] and for the counting of radioactivity. The other part was andlysed for total phosphate by an automated phosphate determination [5, 131. The reagents for the phosphate determination were modified as follows: solution A: 0.36 M HzS04, 31 mM ammonium heptamolybdate and 1 ml Liqui-Nox/l ; solution B: 0.36 M HzS04, 68 mM L-ascorbic acid, 15 mM potassium antimony(I1I)tartrate and l ml Liqui-Nox/I.…”

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

“…The glycosomes in T. brucei contain at least nine enzymes involved in or related to glycolysis, which can account for the conversion of glucose to 3-phosphoglycerate plus glycerol. They are uniquely located inside the glycosomes and therefore exhibit high latency in cell-free extracts (1,7). These organelles also contain part of the adenylate kinase activity (8), two enzymes of pyrimidine biosynthesis (9), and, in insect forms (cultured procyclics), malate dehydrogenase (8,10) and phosphoenolpyruvate carboxykinase (10) form other important metabolic functions in addition to glycolysis.…”