A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22°C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 ± 0.09 µM and a Vmax of 0.65 ± 0.08 pmol·(107 cells)-1·s-1. [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 ± 15 µM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 ± 0.4 µM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.Key words: uracil, trypanosome, proton-nucleobase cotransport, nucleobase transport.

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A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport

Biochemistry and Cell Biology ◽

10.1139/bcb-76-5-853 ◽

1998 ◽

Vol 76 (5) ◽

pp. 853-858 ◽

Cited By ~ 7

Author(s):

Harry P. de Koning ◽

Simon M. Jarvis

Keyword(s):

Trypanosoma Brucei ◽

Trypanosoma Brucei Brucei ◽

High Affinity ◽

Dependent Transport

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Sequence differences between histones of procyclic Trypanosoma brucei brucei and higher eukaryotes

Parasitology ◽

10.1017/s003118200007373x ◽

1992 ◽

Vol 105 (1) ◽

pp. 97-104 ◽

Cited By ~ 26

Author(s):

K. Bender ◽

B. Betschart ◽

J. Schaller ◽

U. Kämpfer ◽

H. Hecker

Keyword(s):

Amino Acid ◽

Trypanosoma Brucei ◽

Protozoan Parasite ◽

Tetrahymena Pyriformis ◽

Reversed Phase ◽

Alternative Methods ◽

Trypanosoma Brucei Brucei ◽

Calf Thymus ◽

Core Histones ◽

Higher Eukaryotes

Four histones, a, b, c, d from procyclic Trypanosoma brucei brucei, which show similarities with the amino acid composition of the core histones H3, H2A, H2B and H4, were isolated and cleaved with Endoproteinase Glu-C. The fragments were separated by FPLC reversed phase chromatography and a subset of the fragments (a5, a9, b6, c8, d3, d9, d11) was subjected to sequence analysis. A 54–71% identity was found in the sequences of the fragment c8 and the C-terminal half of H2B and of three fragments of protein d covering the N-terminal half as well as the C-terminal region of H4. The amino acid sequence of the fragment a9 showed a 57 and 54% identity with H3 sequences of Saccharomyces cerevisiae and Xenopus laevis. Neither the a5 nor the b6 sequence could be aligned with histone sequences of other eukaryotes. The significant differences of 21–48% between the T. b. brucei, histone sequences and those of calf thymus histones, which are more pronounced than the differences of Tetrahymena pyriformis and the higher eukaryote, resulted partially from replacements of amino acids with different properties and indicate specific patterns of histone–histone and/or histone–DNA contact sites in the nucleosome of T. b. brucei. These differences, together with the lack of a functional histone H1, may be sufficient to explain the lack of a salt-dependent formation of the nucleosome filament into the 30 nm fibre, which reflects alternative methods of organizing and processing the genetic information in the nucleus of the protozoan parasite and which may be of chemotherapeutic significance.

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Active transport of l-proline in the protozoan parasite Trypanosoma brucei brucei

Biochemical Journal ◽

10.1042/bj2910297 ◽

1993 ◽

Vol 291 (1) ◽

pp. 297-301 ◽

Cited By ~ 21

Author(s):

C L'Hostis ◽

M Geindre ◽

J Deshusses

Keyword(s):

Trypanosoma Brucei ◽

Membrane Vesicles ◽

Protozoan Parasite ◽

Protonmotive Force ◽

Atpase Inhibitor ◽

Plasma Membrane Vesicles ◽

Proline Transport ◽

Carbonyl Cyanide ◽

Uptake Activity ◽

Filtration Technique

The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (‘CCCP’) did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.

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Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽

10.12688/f1000research.10342.2 ◽

2017 ◽

Vol 6 ◽

pp. 683 ◽

Cited By ~ 31

Author(s):

Terry K. Smith ◽

Frédéric Bringaud ◽

Derek P. Nolan ◽

Luisa M. Figueiredo

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protozoan Parasite ◽

Tsetse Fly ◽

Metabolic Reprogramming ◽

Mammalian Host ◽

Insect Vector ◽

Environmental Signals ◽

Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

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