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

1993 ◽  
Vol 291 (1) ◽  
pp. 297-301 ◽  
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.

scholarly journals Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes

1981 ◽  
Vol 198 (3) ◽  
pp. 535-541 ◽  
Author(s):  
F Mayor ◽  
J G Marvizón ◽  
M C Aragón ◽  
C Gimenez ◽  
F Valdivieso
Keyword(s):  
Rat Brain ◽  
Membrane Vesicles ◽  
Uptake System ◽  
Plasma Membrane Vesicles ◽  
Glycine Uptake ◽  
High Affinity ◽  
Carbonyl Cyanide ◽  
Brain Synaptosomes ◽  
Glycine Transport ◽  
Sole Energy

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.

scholarly journals Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei

1994 ◽  
Vol 304 (1) ◽  
pp. 227-233 ◽  
Author(s):  
A E Vercesi ◽  
S N Moreno ◽  
R Docampo
Keyword(s):  
Plasma Membrane ◽  
Acridine Orange ◽  
Trypanosoma Brucei ◽  
Initial Rate ◽  
Sodium Orthovanadate ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Permeabilized Cells ◽  
Carbonyl Cyanide ◽  
Atpase System

The use of digitonin to permeabilize the plasma membrane of Trypanosoma brucei procyclic and bloodstream trypomastigotes allowed the identification of a non-mitochondrial nigericin-sensitive Ca2+ compartment. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to cause Ca2+ release from this compartment, which was also sensitive to sodium orthovanadate. Preincubation of the cells with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 greatly reduced the nigericin-sensitive Ca2+ compartment. Bafilomycin A1 inhibited the initial rate of ATP-dependent non-mitochondrial Ca2+ uptake and stimulated the initial rate of nigericin-induced Ca2+ release by permeabilized procyclic trypomastigotes. ATP-dependent and bafilomycin A1- and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl)-sensitive Acridine Orange uptake was demonstrated in permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles by a process inhibited by bafilomycin A1, NBD-Cl, nigericin, and Ca2+. Vanadate or EGTA significantly increased Acridine Orange uptake, while Ca2+ released Acridine Orange from these preparations, thus suggesting that the dye and Ca2+ were being accumulated in the same acidic vacuole. Acridine Orange uptake was reversed by nigericin, bafilomycin A1 and NH4Cl. The results are consistent with the presence of a Ca2+/H(+)-ATPase system pumping Ca2+ into an acidic vacuole, that we tentatively named the acidocalcisome.

scholarly journals A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei; selective inhibition by pentamidine

1993 ◽  
Vol 296 (3) ◽  
pp. 759-763 ◽  
Author(s):  
G Benaim ◽  
C Lopez-Estraño ◽  
R Docampo ◽  
S N J Moreno
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Membrane Vesicles ◽  
Selective Inhibition ◽  
Fine Tuning ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Calmodulin Antagonist ◽  
Stimulation Of

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax. of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km,app. for Ca2+ was 0.52 +/- 0.03 microM. This was decreased to 0.23 +/- 0.05 microM, and the Vmax. was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca(2+)-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca(2+)-ATPase by calmodulin.

Na(+)-dependent and Na(+)-independent systems of choline transport by plasma membrane vesicles of A549 cell line

1994 ◽  
Vol 267 (5) ◽  
pp. C1279-C1287 ◽  
Author(s):  
A. Kleinzeller ◽  
C. Dodia ◽  
A. Chander ◽  
A. B. Fisher
Keyword(s):  
A549 Cells ◽  
Membrane Vesicles ◽  
Choline Uptake ◽  
Plasma Membrane Vesicles ◽  
Choline Transport ◽  
High Affinity ◽  
A Value ◽  
Carbonyl Cyanide ◽  
Rate Limiting ◽  
A549 Lung

Membrane vesicles of A549 lung cells accumulate choline by two pathways: the Na(+)-independent uphill uptake of choline [Michaelis-Menten constant (Km) approximately 44 microM; steady-state gradient approximately 45 at 5 microM external choline] is dependent on a transmembrane H+ gradient, is relatively insensitive to hemicholinium-3, is amiloride sensitive, and is abolished by valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The Na(+)-dependent active choline uptake (Km approximately 4 microM, inhibitor constant for hemicholinium-3 approximately 0.1 microM), is specific for Na+, is amiloride and FCCP sensitive, and is electrogenic: the overshoot using K(+)-loaded vesicles and NaCl gradient was increased by valinomycin. The time of the overshoot peak, T was approximately 90 s in a NaSCN medium (or in presence of other lipid-soluble anions), a value close to that for alpha-aminoisobutyrate as substrate (T = approximately 1.5 min). T was lengthened in NaCl medium to approximately 10 min, and the overshoot was abolished by impermeant anions. External Cl- is not required for the choline uptake: valinomycin produced an overshoot in the presence of only impermeant anions, with T approximately 90 s. Most of the above properties are shared by the high-affinity Na(+)-dependent choline transport in synaptosomes. The characteristics of the Na(+)-dependent choline uptake by membrane vesicles of A549 cells are consistent with an electrogenic choline(+)-Na+ cotransport, with the rate-limiting anion (e.g., Cl-) influx balancing the positive charges transferred into the vesicles. The data are also consistent with an involvement of an amiloride-sensitive choline+/H+ antiport (or choline(+)-OH- symport) in the low- and high-affinity choline uptake pathways.

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

1998 ◽  
Vol 76 (5) ◽  
pp. 853-858 ◽  
Author(s):  
Harry P de Koning ◽  
Simon M Jarvis
Keyword(s):  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Trypanosoma Brucei Brucei ◽  
Uptake Mechanism ◽  
Pyrimidine Nucleosides ◽  
High Affinity ◽  
Carbonyl Cyanide ◽  
Dependent Transport ◽  
Extracellular Sodium ◽  
Sodium And Potassium

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.

scholarly journals Protonmotive-force-driven leucine uptake in yeast plasma membrane vesicles

FEBS Letters ◽  
1987 ◽  
Vol 213 (1) ◽  
pp. 45-48 ◽  
Author(s):  
Miroslava Opekarová ◽  
Arnold J.M. Driessen ◽  
Wil N. Konings
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Protonmotive Force ◽  
Plasma Membrane Vesicles ◽  
Yeast Plasma Membrane ◽  
Leucine Uptake

Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1

2007 ◽  
Vol 292 (2) ◽  
pp. F593-F598 ◽  
Author(s):  
Masahiro Tsuda ◽  
Tomohiro Terada ◽  
Jun-ichi Asaka ◽  
Miki Ueba ◽  
Toshiya Katsura ◽  
...  
Keyword(s):  
Driving Force ◽  
Brush Border Membrane ◽  
Organic Cation ◽  
Functional Characterization ◽  
Membrane Vesicles ◽  
Hek293 Cells ◽  
Plasma Membrane Vesicles ◽  
Renal Brush Border Membrane ◽  
Carbonyl Cyanide ◽  
Overshoot Phenomenon

Recently, we have isolated the rat (r) H+/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H+ gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [14C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [14C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H+ gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [14C]tetraethylammonium uptake by an H+ gradient [intravesicular H+ concentration ([H+]in) > extravesicular H+ concentration ([H+]out)] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [14C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H+ gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.

scholarly journals Sodium-dependent alanine transport in plasma-membrane vesicles from rat liver

1978 ◽  
Vol 174 (3) ◽  
pp. 1083-1086 ◽  
Author(s):  
J M M van Amelsvoort ◽  
H J Sips ◽  
K van Dam
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Alanine Transport ◽  
Carbonyl Cyanide ◽  
Sodium Dependent

L-Alanine transport was studied in plasma-membrane vesicles from rat liver. A gradient of NaSCN, but not of KSCN, stimulated alanine uptake. Monensin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished the observed overshoot in uptake. After equilibration of alanine, NaSCN induced uphill transport.

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