Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14 C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms : firstly by a facilitated-diffusion carrier showing a K m of 0.17 mM and a Vmax of 44 nmol 10 Ϫ8 cells min Ϫ1 that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton-motive-force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74% decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T. brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.Keywords : glycerol uptake; facilitated diffusion; inhibitor studies; Trypanosoma brucei.Trypanosoma brucei, a protozoan parasite which causes sleeping sickness and economically important cattle diseases in Africa, is transmitted by the tsetse-fly. Insect and bloodstream forms show marked biochemical differences, especially with respect to energy metabolism. While the insect form contains a well-developed mitochondrion with both a functional citric acid cycle and respiratory chain, bloodstream forms rely exclusively on glycolysis for their ATP production [1,2]. Interestingly, in all members of the order Kinetoplastida, glycolysis occurs in a characteristic organelle: the glycosome [3]. Under anaerobic conditions, e.g. induced by salicyl hydroxamate which inhibits glycerol-3-phosphate oxidase, bloodstream-form trypanosomes produce equimolar amounts of pyruvate and glycerol [4]. At high concentrations both metabolites are toxic for the parasite and have to be removed in order to avoid cell death. Since a specific pyruvate transporter was recently demonstrated in T. brucei [5,6], we were interested in how glycerol transport across the plasma membrane occurs. The existence of a specific glycerol carrier in bloodstream-form trypanosomes would offer the possibility to interfere effectively with the parasite's metabolism Correspondence to M. Duszenko, Physiologisch-chemisches Institut,
The glucose transporter of Trypanosoma hrucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (K," = 80 pM, V,,, = 4 nmol min-' lo-' cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Kl = 21 mM). Control experiments measuring deoxy-~-['H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Qlo = 2.33 and an activation energy of E, = 64 kJ mol-'. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 pM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 pM), the ionophor monensin (1 pM), or the Na'/K'-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na'-free conditions in the absence of a proton gradient, the specific activity of D-['4C]glUCoSe transport was determined as 2.9 nmol min-' (mg protein)-' at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.Keywords: facilitated diffusion; functional reconstitution ; glucose uptake ; hexose transporter; Trypanosoma brucei.African trypanosomes cause severe sleeping sickness in human and nagana in livestock throughout the equatorial part of the continent, threatening millions of people and vastly affecting meat and diary production. This protozoan parasite undergoes a complex lifecycle, including distinct stages in both the mammalian host and the insect vector. Vector and bloodstream forms show marked differences, especially in energy metabolism, which is restricted to glycolysis in the latter forms, whereas insect forms possess a well developed mitochondrion and express a functional citric acid cycle as well as the respiratory chain. Hence, glucose uptake is pivotal to bloodstream-form metabolism but seems only marginally important for procyclic vector forms. Nevertheless, procyclic-form parasites metabolize glucose from the medium and we thus questioned whether the respective glucose transporter is different from that expressed in bloodstream forms, i.e. if expression of the glucose carrier is differentially regulated during the lifecycle. This question seemed intriguing, since conflicting results have been published previously. Whereas earlier data suggested that glucose uptake ...
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