Active transport of 2-deoxy-d-glucose in Trypanosoma brucei procyclic forms

Molecular and Biochemical Parasitology, 42 (1990) 197-204

Elsevier

MOLBIO 01388

197

Active transport of 2-deoxy-D-glucose in Trypanosoma brucei procyclic forms

Mari lyn Parsons 1'2 and Barbara Nielsen 1

1Seattle Biomedical Research Institute, Seattle, WA and 2Department of Pathobiology, University of Washington, Seattle, WA, U.S.4.

(Received 6 February 1990; accepted 30 April 1990)

The characteristics of glucose transport by procyclic forms of Trypanosoma brucei were examined in a rapid transport assay using the glucose analogue 2-deoxyglucose. In contrast to bloodforms where the Km for 2-deoxyglucose transport is about 1 mM, procyclic forms have a Km of about 38 #M. Procyclic forms show temperature-dependent, saturable import, and import of 2-deoxyglucose is competitive with glucose and mannose. Unlike the bloodforms which employ facilitated diffusion, the procyclic forms actively transport glucose. Use of inhibitors and ionophores suggests that a protonmotive force is required for glucose transport in procyclic forms. Unlike the human erythrocyte glucose transporter, the glucose transporter of the T. brucei procyclic form is relatively insensitive to inhibition by cytocholasin B.

Key words: Trypanosoma brucei; Procyclic form; Glucose transporter; 2-Deoxyglucose; Kinetics

Introduction

Trypanosoma brucei is a protozoan parasite which relies on host molecules as energy sources. During the life cycle, T. brucei lives alternately in the bloodstream of mammalian hosts, bathed in homeostatically maintained 5 mM glucose, and in the digestive tracts of tsetse flies. The bloodforms rely completely on the external glucose for energy metabolism via glycolysis, and lyse within 10 min of glucose starvation [1]. The essential nature of glucose transport in bloodforms suggests that it might be a suitable target for chemotherapeutic intervention, should the transporter be significantly different from the host transporters. Recent work indicates that this is indeed the case [2]. Procyclic culture forms, homologous to the insect midgut stage, can thrive quite happily in the absence of glucose, preferring instead amino acids as energy sources [3]. These characteristics suggested that glucose transport might be stage-regulated.

Early work on bloodforms demonstrated high levels of glucose transport, with a Km of approximately 1.2 mM [1,4]. On the other hand, procyclic forms were found to transport little glucose [5]. In the latter study, however, a 2-h assay was employed, and under such conditions glycolysis, which is also stage-regulated [6], could have a significant impact on driving the uptake of glucose. More recently, other workers have synthesized the non-metabolizable substrate, 6-deoxyglucose, and have demonstrated that glucose transport in trypanosome bloodforms occurs by carrier-mediated facilitated diffusion and is the rate-limiting step in glucose metabolism [2]. We have developed a rapid assay to measure the import of (com- mercially available) 2-deoxy-D-glucose (2DOG). Our data indicate that the glucose transporters in the two life cycle stages have distinct properties, and that these properties are significantly different from the best characterized human glucose transporter, the erythrocyte facilitative carrier.

Correspondence address: M. Parsons, Seattle Biomedical Re- search Institute, 4 Nickerson Street, Seattle, WA 98109, U.S.A.

Abbreviations: 2DOG, 2-deoxy-D-glucose; DCCD, N',N/- dicyclohexylcarbodiimide; FCCP, carbonyicyanide p-(trifluoromethoxy) phenyihydrazone.

Materials and Methods

Trypanosomes. Procyclic forms of EATRO 164 clone IHRI 1 [7] were grown in SDM-79 medium

0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)

198

[8] and harvested in early to mid-log phase (5 × 106 - 10 7 ml- l ) . Bloodforms were grown in intact rats, harvested in Cunningham's SM-77 medium [9] supplemented with 5% serum and 10 mM glucose, and purified by DEAE chromatography [10].

Transport assay. Procyclic trypanosomes were rinsed twice in SM-77 medium, pH 6.8, lack- ing sugars, proline, and organic acids before the assay. Because glucose is rapidly metabolized, this insured that the assay was performed under zero-trans conditions (with no substrate inside the cells). Procyclic cells maintained viability for sev- eral hours in the test medium. We have used a modification of the rapid transport assays which were developed for studying nucleoside transport in mammalian cells [ 11] and in Leishmania [ 12]. Assays were performed in triplicate or quadrupli- cate. Cells (108 m1-1) were diluted with an equal volume of the same medium containing radioac- tive 1 #Ci [1,2-3H] 2DOG (15-50 Ci mmo1-1, NEN) plus various concentrations of sugars or inhibitors. The cells (107 in 200 #1) were removed from the substrate by centrifugation through 100 #1 of a mixture of 95% dibutyl phthalate (Sigma), 5% paraffin oil (Fisher). Both 6-s and 30-s in- cubations at room temperature (23°C) were used unless otherwise noted. The aqueous supernatant was removed, the interface rinsed twice, and the oil was then removed. The cell pellets were lysed in 100 Izl of 1% Triton X-100 and the radioactivity determined by scintillation counting. Non-specific trapping of radiolabel was estimated by immedi- ate centrifugation of cells through assay medium containing 10 mM 2DOG.

The Km of 2DOG transport in procyclic forms was determined by a Hanes plot (substrate concentration versus the substrate/velocity). Both 6-s and 30-s measurements were made, with no significant differences noted between the two time points. Substrate concentrations ranging from 1 /zM to 1 mM were examined.

Cellular protein was determined for each cell preparation using the bicinchoninic acid method (Pierce). 107 procyclic cells were estimated to contain 55 #g protein by this procedure. Inhibitors and sugars were purchased from Sigma.

Quantitation of phosphorylation. The metabolism of 2DOG was arrested following the assay by lysing the cells with 5.5% perchloric acid. Fol- lowing neutralization with 5% KOH, duplicate aliquots were counted directly. Similarly, duplicate aliquots were precipitated with the Somo- gyi reagent which precipitates phosphorylated glucose derivatives [13,14], in this case 2DOG-6- phosphate, and the radioactivity remaining in the supernatant was determined.

Results

Glucose transport in trypanosome bloodforms is carrier mediated, and proceeds with a Km of about 1 mM and a Vmax of approximately 29 nmol min -1 per 108 cells [2]. Little incorporation of radiolabel was observed in procyclic forms using millimolar concentrations of glucose [ 15]. 2DOG is a glucose analogue (modified at the C-2 position) and is an efficient substrate for the glucose transporters of many cells, including trypanosome bloodforms [ 1]. 2DOG can be phosphorylated by hexokinase, but cannot be metabolized further. As shown in Fig. 1, transport of [3H]glucose by procyclic cells is inhibited by 2DOG. Little radiolabel is accumulated at high 2DOG concentrations in this 30-s assay; however at low 2DOG concentrations accumulation of radiolabel is high (> 15 000 cpm per 107 cells). The transport of these substrates is saturable, reaching in this case approximately 7 nmol min-I (mg protein) -~. 1 mM L- glucose does not inhibit the transport of D-glucose (see inset), indicating that transport is stereospe- cific. The K~ for 2DOG inhibition of glucose transport and the Km for 2DOG transport (see below) were both calculated to be 38 #M, indicating that glucose and 2DOG probably share the same transport system in procyclic forms. Therefore 2DOG was used to study the process of glucose transport in these organisms.

Fig. 2 shows the time course for 2DOG uptake in procyclic forms at 50 #M 2DOG. These experiments demonstrate that the uptake of the radiolabel is linear over approximately 5 min (the inset shows an expanded view of the first 90 s), and that equilibrium is reached within 10-15 min. At 4°C, little transport was seen (not shown).

199

20 7

1 6 ~ - - -

_ _ ~ _ 5

~¢-- 3 , " | "

o 2 ~

C

lie L-(I 2 . ~ , 1

o , , , , , , , , , ~ - - t . . ~ , , ~ , o 0 100 200 300 400 500 900 1000 1100

2DOG (uM)

Fig. 1. [3H]-D-Glucose transport by T. brucei procyclic forms. Incorporation of radiolabelled glucose was measured in a 30- s assay, with the addition of various concentrations of 2DOG. The effect of L-glucose on glucose transport is shown in the

inset. NI, no inhibitor;, L-G, 1 mM L-glucose; 2DOG, 1 mM 2DOG.

701

eo

4O

2 0

1 0

/ 2L/ °¢~ - - ~ ~ - --~o

0 5 10 15 2 0 25 30 35

t ime (rain)

Fig. 2. Time course for 2DOG uptake by T. brucei procyclic forms. Procyclic forms were incubated with 50/,M 2DOG for the indicated times at room temperature (23°C) and removed from the substrate by centrifugation through inert oil. The inset

shows a 90-s time course.

After 30 min incubation with 50 #m 2DOG, there was 3.2 nmol of 2DOG within 107 procyclic cells, while 6.8 nmol remained in the external medium (200 #1). Using the water space of trypanosome procyclic cells estimated by Ghiotto et al. [16], the volume of 107 cells is 0.54/zl, and therefore the radiolabel is 185-fold more concen- trated within the cells. The proportion of 2DOG

which was phosphorylated at 30 min was determined by precipitation with Somogyi reagent to be 77%. Thus a 43-fold concentration gradient of 2DOG between the external and intracellular 2DOG exists, assuming that all of the water space is available to the 2DOG. These findings demonstrate that the procyclic trypanosome is capable of transporting glucose against a concentration gradient, a characteristic of active transport.

Km was estimated to be 38 /zM and Vmax was 9.8 nmol min-1 (mg protein) - l . No evidence of a second transporter system with a high Kin, like that of bloodforms, was seen using high 2DOG concentrations (or when the data presented in Fig. 1 was replotted). The Vmax measurements varied up to three-fold depending on the cell preparation used, suggesting that the level of transporter may fluctuate.

The specificities of the procyclic transporter were determined by measuring the inhibition of 2DOG transport well below Km (1 #M), where the Ki can be estimated by plotting the inhibitor concentration versus V0/V. The X-intercept then represents -Ki [2]. Table I lists the inhibitor constants found for various sugars and analogues with the procyclic transporter. Glucose, mannose, and

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TABLE I Inhibition constants (K0 for glucose analogues

2-Deoxyglucose a 38 ItM Glucose 45 #M Mannose 31 ,uM Galactose 0.59 mM Fructose 1.5 mM Glucosamine 2.0 mM 3-O-methyl glucose 5.1 mM a-Methyl mannoside >20 mM Phloridzin 1.1 mM

a K m

2DOG all interact with similar, high affinities. Fructose, galactose, and glucosamine have approximately 20-fold lower affinities than glucose does. Finally, 3-O-methyl glucose and c~-methyl- mannoside show very little inhibition of the procyclic transporter. We have found that phloridzin (glucose which is derivatized with phloretin at the C-1 position, in the/3 configuration) is an inhibitor of glucose transport in procyclic forms with a Ki of 1.1 mM.

Active transport of glucose usually involves cotransport of an ion down its concentration gradient to provide energy [17]. The high concentrations of phloridzin (a potent inhibitor of Na+-coupled glucose transport, Ki < 10/zM [18]) required to inhibit glucose transport in procyclic forms suggests that Na ÷ is not the counterion. This was corrobo- rated by finding that glucose transport was similar in sodium-free buffer (0.1 M KPO4, pH 7.0) and in buffer containing 20 mM NaCI (not shown).

In the related kinetoplastid Leishmania donovani, high affinity (Km 24 /zM) glucose transport is associated with H ÷ symport, which in turn depends on the active maintenance of a proton gradient [19]. Therefore we examined the effect of compounds which inhibit glucose transport in Leishmania, such as respiratory inhibitors and ionophores. As shown in Fig. 3, the respiratory inhibitor KCN (used at 2 mM, to inhibit both the SHAM-sensitive and insensitive terminal oxidases; ref. 20) was an effec- tive inhibitor 2DOG transport by procyclic forms. Oligomycin, which inhibits mitochondrial AT- Pases, partially inhibited 2DOG transport at 1 #g m1-1. The plasma membrane ATPase inhibitor N, N'-dicyclohexylcarbodiimide (DCCD) showed

1 0 0

8O A o

s o

4 0

2 0

0

DCCD OL KCN VAL NIG V ÷ N FCCP inhibitor

Fig. 3. Effects of respiratory inhibitors and ionophores on 2DOG transport. Inhibitors and ionophores were preincubated with procyclic cells, which were then tested for their abil- ity to transport 2DOG by the addition of an equal volume of medium containing 60 #M 2DOG. Transport after preincubation with these compounds was compared to transport after preincubation with solvent alone. Both 6-s and 30-s assays were performed, with no significant differences. Inhibitor concentrations and preincubation times are as follows: DCCD, 1 raM, 10 min; OL, oligomycin, 1 #g ml - j , 10 min; KCN, 2 mM, 10 min; VAL, valinomycin, 1 #M, 2 min; NIG, nigericin, 3 ~M, 2 min, V+N: valinomycin, 1 #M, plus nigericin, 3 #M,

2 min; FCCP: 5 #M, 10 rain.

partial inhibition, but required relatively high concentrations. The K ÷ ionophore valinomycin, and nigericin, a K+/H + antiporter, both partially inhibited transport at low concentrations. Com- bined together these compounds inhibited transport more effectively. Finally, carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a proton ionophore, blocked 80% of glucose transport at 5 #M. The pattern of inhibition shown is similar indeed to that seen L. donovani [19].

Other classic inhibitors of glucose transport were also examined (Table II). N-Ethylmaleimide, a permeant sulfhydryl reagent, and p-hydroxymercuribenzoate, a non-permeant sulfhydryl reagent, both inhibit procyclic form glucose transport. This data suggests that an exofacial sulfhydryl group may be important for activity of the transporter. Papaverine, an inhibitor of glucose transport in mammalian cells with a Ki of 130/zM [21], partially inhibited 2DOG transport in procyclic forms, but at much higher concentrations (1 mM). Similar inhibition of transport in bloodforms was observed (unpublished). Cytocholasin B is a potent inhibitor of the human erythrocyte transporter with a Ki of approximately 0.5 #M

TABLE II Glucose transporter inhibitors

% control 2DOG transport a

N-Ethylmaleimide, 1 mM 2 min 53

10 min 12

p-hydroxymercuribenzoate, 1 mM 2 min 57

10 min 17

Papaverine, 10 min 100/~M 87

1 mM 43

Cytocholasin B, 10 min 10 #M 80

100 #M 41

aThe effects of inhibitors on 2DOG transport was measured as described in Fig. 3. Inhibitor concentration and preincubation times are indicated.

[22]. 100 #M cytocholasin B was required to inhibit 50% of 2DOG transport in procyclic forms.

Discussion

In this communication we demonstrate the ex- istence of a high affinity glucose transporter in T. brucei procyclic forms. This transporter actively transports 2DOG, with a Km of about 38/~m and a Vma~ of about 9.8 nmol min-~ (mg protein)- or 5.4 nmol min - l per 108 cells. This is about 1/6 the Vm~x of 6-deoxyglucose transport at 20°C of bloodforms on a per cell basis. Nevertheless, the rate of glucose transport by procyclic cells is sufficient for their survival with glucose as the sole energy source for at least 2 h (unpublished observations).

No evidence of a second, high-Kin transport system was seen in procyclic forms (using either glucose or 2DOG as substrates), indicating that the facilitated diffusion transporter of bloodforms is expressed in a stage-regulated manner. Whether slender bloodforms express the low-Kin transporter, in addition to the high Km transporter is not clear. However, we have found that transport of 2DOG by slender bloodforms is not inhibited by FCCP at substrate concentrations close

201

to the Kms of either transporter (unpublished re- suits). No data exist on the kinetics of glucose transport in intermediate and stumpy bloodforms. These questions are of potential significance when one considers the possibility of developing drugs directed against the glucose transporter of pathogenic bloodforms.

The specificity of the high affinity procyclic transporter observed appears similar, but not iden- tical, to that of the low affinity bloodform transporter (as measured using 6-deoxyglucose). How- ever, galactose does not inhibit 6-deoxyglucose transport in bloodforms. This suggests that hydro- gen binding by the hydroxyl at the C-4 position, which is postulated to be required for binding to the bloodform transporter [2], is much less important for interaction with the high affinity transporter. The specificities of the trypanosome glucose transporters are quite different from the human erythrocyte transporter, which accepts substi- tutions at the C-3 (i.e., 3-O-methyl glucose) and C-1 (a-D-glucose) positions. The unique specificities of the trypanosome transporters suggest that they may make good candidates for chemotherapeutic intervention. However, biochemical and molecular cloning studies have revealed that hu- mans possess a diverse group of glucose transporters, both of the facilitated diffusion and the Na ÷ cotransport type [23-28]. The specificities and characteristics of some of these transporters have not been well studied. Therefore, the effects of potential inhibitors of trypanosome glucose transport on all of the human (and other host) transporters will have to be considered.

The characteristics of glucose transport in T. brucei procyclic forms closely resembles that found in Leishmania promastigotes [19,29]. The strong inhibition of transport by FCCP, and addi- tive inhibition by valinomycin and nigericin observed in T. brucei procyclic forms was observed in Leishmania promastigotes also. There, active transport driven by a protonmotive force has been demonstrated [ 19]. It is interesting to note that the two parasites in these particular stages reside in similar environments, i.e., the fly gut. As in other cases where glucose transport occurs against a concentration gradient [ 17], the trypanosome procyclic transporter is probably a cotransporter. We hypothesize on the basis of the data using vari-

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ous inhibitors, in particular the proton ionophore FCCP, that H ÷ is the counterion. Recently it has been demonstrated that the intraerythrocytic forms of Plasmodium yoelii actively transport glucose across the parasite membrane and it was suggested that this too was driven by protonmotive force [30]. In higher eukaryotes transport of glucose is driven by Na ÷ electrochemical gradients or occurs by facilitated diffusion, while in bacteria it is mediated by binding proteins (the permease systems) or is driven by proton gradients [17]. Thus the Kinetoplastida appear to occupy a pivotal position in the evolution of glucose transporters, possess- ing both a facilitated diffusion glucose transporter and a glucose/H ÷ cotransporter. It will be interesting to examine the genes for these transporters and compare their evolutionary position to the human erythrocyte transporter and the Escherichia coli xylose/H ÷ and arabinose/H ÷ cotransporters which show significant homology at the amino acid level [311.

Acknowledgements

The authors thank Drs. Dan Zilberstein, Keith Alexander and Elizabetta Ullu for helpful discus- sions. We also thank Victoria Carter for tech- nical assistance and Koral Massie-Lavelle and Karen Kinch for preparing the manuscript. This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, the National Institutes of Health (AI22635) and the Murdock Charitable Trust.

References

1 Gruenberg, J., Sharma, P.R. and Deshusses, J. (1978) D- Glucose transport in Trypanosoma brucei: D-glucose transport is the rate-limiting step of its metabolism. Eur. J. Biochem. 89, 461-469.

2 Eisenthal, R., Game, S. and Hoiman, G.D. (1989) Speci- ficity and kinetics of hexose transport in Trypanosoma brucei. Biochim. Biophys. Acta 985, 81-89.

3 Vickerman, K. (1985) Developmental cycles and biology of pathogenic trypanosomes. Br. Med. Bull. 41, 105-114.

4 Southworth, G.C. and Read, C.P. (1970) Specificity of sugar transport in Trypanosoma garnbiense. J. Protozool. 17, 396-399.

5 Von Brand, T., Tobie, E.J. and Higgins, H. (1967) Hex- ose and glycerol absorption by some Trypanosomatidae. J. Protozooi. 14, 8-14.

60pperdoes, F.R. (! 987) Compartmentation of carbohydrate

metabolism in trypanosomes. Annu. Rev. Microbiol. 41, 128-151.

7 Stuart, K., Gobright, E., Jenni, L., Milhausen, M, Thomashow, L.S. and Agabian, N. (1984) The IsTaR serodeme of Trypanosoma brucei: development of a new serodeme. Parasitology 70, 747-754.

8 Brun, R. and Schononberger, M. (1979) Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop. 36, 289-292.

9 Cunningham, I. (1977) New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325-329.

10 Lanham, S.M. (1968) Separation of trypanosomes from the blood of infected rats and mice by anion-exchangers. Nature 218, 1273-1274.

!1 Aronow, B., Allen, K., Patrick, J. and Ullman, B. (1985) Altered nucleoside transporters in mammalian cells se- lected for resistance to the physiological effects of inhibitors of nucleoside transport. J. Biol. Chem. 260, 6226-6233.

12 Aronow, B., Kaur, K., McCartan, K. and Ullman, B. (1987) Two high affinity nucleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22, 29-37.

13 Kletzien, R.F. and Perdue, J.F. (1973) The inhibition of sugar transport in chick embryo fibroblasts by cytocholasin B. Evidence for a membrane-specific effect. J. Biol. Chem. 248, 711-719.

14 Somogyi, M. (1945) Determination of blood sugar. J. Biol. Chem. 160, 69-73.

15 Parsons, M., Alexander, K., Hill, T., Nielsen, B., Dovey, H.F. and Wang, C.C. (1990) Glucose import and glyco- somal biogenesis in Trypanosoma brucei. In: Parasites: Molecular Biology, Drug and Vaccine Design (Agabian, N. and Cerami, A., eds.) pp.247-261, Wiley-Liss, New York.

16 Ghiotto, V., Brun, R., Jenni, L. and Hecker, H. (1979) Try- panosoma brucei. morphometric changes and loss of infec- tivity during transformation of bloodstream forms to procyclic culture forms in vitro. Exp. Parasitol. 48, 447-456.

17 Baly, D.L. and Horuk, R. (1988) The biology and bio- chemistry of the glucose transporter. Biochim. Biophys. Acta 947, 571-590.

18 Turner, R.J. and Silverman, M. (1977) Sugar uptake into brush border vesicles from normal human kidney. Proc. Natl. Acad. Sci. USA 74, 2825-2829.

19 Zilberstein, D. and Dwyer, D.M. (1985) Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc. Natl. Acad. Sci. USA 82, 1716-1720.

20 Njogu, R.M., Whittaker, C.J. and Hill, G.C. (1980) Ev- idence for a branched electron transport chain in Try- panosorna brucei. Mol. Biochem. Parasitol. 1, 13-29.

21 Steinfelder, H.J. and Joost, H.G. (1988) Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors. FEBS Lett. 227, 215-219.

22 Taverna, R.D. and Langdon, R.G. (1973) Reversible asso- ciation of cytocholasin B with the human erythrocyte membrane. Inhibition of glucose transport and the stoichiome- try of cytocholasin binding. Biochim. Biophys. Acta 323, 207-219.

23 Fukumoto, H., Seino, S., lmura, H., Seino, Y., Eddy, R.L., Fukushima, Y., Byers, M.G., Shows, T.B. and Bell, G.I. (1988) Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter- like protein. Proc. Natl. Acad. Sci. USA 85, 5434-5438.

24 Sarkar, H.K., Thorens, B., Lodish, H.F. and Kaback, H.R.

(1988) Expression of the human erythrocyte glucose transporter in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5463-5467.

25 Kayano, T., Fukumoto, H., Eddy, R.L., Fan, Y.-S., By- ers, M.G., Shows, T.B. and Bell, G.I. (1988) Evidence for a family of human glucose transporter-like proteins. Se- quence and gene localization of a protein expressed in fe- tal skeletal muscle and other tissues. J. Biol. Chem. 263, 15245-15248.

26 Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Sequence and structure of a human glucose transporter. Science 229, 941-945.

27 Bimbanm, M.J., Haspel, H.C. and Rosen, O.M. (1986) Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc. Natl. Acad. Sci. USA 83, 5784-5788.

203

28 Fukumoto, H., Kayano, T., Buse, J.B., Edwards, Y., Pilch, P.F., Bell, G.I. and Seino, S. (1989) Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin- responsive tissues. J. Biol. Chem. 264, 7776--7779.

29 Zilberstein, D. and Dwyer, D.M. (1984) Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Par- asitol. 12, 327-336.

30 Izumo, A., Tanabe, K., Kato, M., Doi, S., Maekawa, K. and Takada, S. (1989) Transport processes of 2-deoxy-D- glucose in erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. Parasitology 98, 371-379.

31 Maiden, M.C.J., Davis, E.O., Baldwin, S.A., Moore, D.C.M. and Henderson, P.J.F. (1987) Mammalian and bac- terial sugar transport proteins are homologous. Nature 325, 641-643.

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Active transport of 2-deoxy-d-glucose in Trypanosoma brucei procyclic forms