Nucleoside transport in Crithidia luciliae

Inlrmarionul Journal fir Prinred in Greur Brifuin

ParrrJirolog~ Vd 23. No. 8, pp. 1039-104,

NUCLEOSIDE SIMONE School of Biochemistry

1993

TRANSPORT

IN CRITHIDIA

T. HALL, GLENDA A. ODGERS and ANNETTE

& Molecular

Genetics,

LUCILIAE M. GERO*

The University of New South Wales, P.O. Box I, Kensington, Australia

NSW 2033,

(Received IO February 1993; accepted 25 April 1993) Abstract-HALL S.T., ODGERS G.A. and GERO A.M. 1993. Nucleoside transport in Crifhidiu luciliae. Infernational Journal for Parasitology 23: 1039-1044. Nucleoside transport was evaluated in the trypanosomatid Crilhidia luciliue by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent K,,, values for adenosine and guanosine were 9.34 f 1.30and 10.6 f 2.60 ,UM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, L-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.

INDEX KEY WORDS: Adenosine transport; nucleosides; Crifhidiu luciliae; Trypanosomatidae.

INTRODUCTION

Crithidia luciliue, a trypanosomatid parasite of the mosquito, is related to the human parasitic flagellates, trypanosomes (sleeping sickness and Chagas disease) and Leishmania (cutaneous and visceral leishmaniasis). Like all parasitic protozoa studied to date they are unable to synthesize purines de novo and are thus dependent on their host for the salvage of exogenous preformed purines for their reproduction and growth (Marr, Berens & Nelson, 1978; Hammond & Gutteridge, 1984; Sherman, 1984). In order to analyse the process by which purine nucleosides are taken up by the plasma membrane of the parasite C. Iuciliae we have used a rapid sampling technique that permits transport measurements over short time intervals. This has allowed us to investigate transport per se, the process by which purine nucleosides permeate the plasma membrane of the parasite, rather than uptake in which the metabolism of the substrate is also taken into account. Thus, experimentation has been carried out over extremely short (second) time intervals. Nucleoside transport has been studied in a number

*To whom all correspondence

should be addressed.

of parasitic protozoa, such as P. fulciparum malaria (Gati, Stoyke, Gero & Paterson, 1987; Gero, Bugledich, Paterson & Jamieson, 1988; Gero & Upston, 1992), Bubesia bovis (see Gero, 1989), Leishmania donovani promastigotes (Aronow, Kaur, McCartan & Ullman, 1987), Trypunosoma cruzi (see Finley, Cooney & Dvorak, 1988). Trichomonas vaginalis (see Harris, Beechey, Linstead & Barrett, 1988) and Giardiu intestinalis (see Davey, Mayrhofer & Ey, 1992) and in each case has been shown to be significantly different to the transport systems for nucleosides in mammalian cells (Paterson & Cass 1986; Jarvis, 1987). In the present study we have characterised the nucleoside transport system in C. luciliae in some detail based on competition and kinetic studies and have compared it with the nucleoside transport system in related organisms. MATERIALS

AND METHODS

Chemicals. [2-‘H]Adenosine (962 GBq mmol ‘), [5‘Hluridine (1.04 TBq mmol-‘), [6-‘Hlthymidine (0.85 TBq mmol-‘), [8-“Clinosine (2.11 GBq mmolF’), deoxy[6‘Hluridine (614 GBq mmol-‘), [‘H]H,O (37 GBq) and inulin [Vlcarboxylic acid (0.24 GBq mmol-‘) were obtained from Amersham International, Buckinghamshire, U.K. [8-‘H]LAdenosine (1.41 TBq mmol-‘) was obtained from Moravek

1039

1040

S. T. HALL et al.

Biochemicals Inc., California, U.S.A. [8-‘H]Guanosine (0.13 TBq mmol ‘) was obtained from Sigma Chemical Co., St. Louis, MO. Deoxy[8-‘Hladenosine (0.48 TBq mmol- ‘), deoxy[8-3H]guanosine (0.33 TBq mmol ‘), deoxy[5-‘Hlcytidine (0.74 TBq mmol-‘) were obtained from ICN Radiochemicals, Division of ICN Biomedicals Inc., California, U.S.A. NBMPR (nitrobenzylthioinosine) (6-[4-nitrobenzyl). thio]-9-po-ribo-furanosylpurine), tubercidin (‘l-deazaadenosine), sangivamycin (7-deaza-7-amido-adenosine), valinomycin, nigericin, p-hydroxymercuribenzoate (pHMB), p-chloromercuribenzoate sulphonate @-CMBS), Nethylmaleimide, ouabain and dipyridamole were purchased from Sigma Chemical Company, St. Louis, MO., U.S.A. Dilazep was a gift from Dr J. Wiley, Austin Hospital, Heidelberg, Victoria, Australia. cx-Aminooxyacetic acid was obtained from Calbiochem-Behring Corp. (La Jolla, CA, U.S.A.). Di-n-butyl phthalate and di-iso-octyl phthalate were from Ajax Chemicals (Blacktown, N.S.W., Australia). An oil mix of a density of 1.03 g ml ’ was prepared by mixing eight parts di-n-butyl phthalate to two parts di-iso-octyl phthalate. A saturated solution of NBMPR was obtained by stirring in PBS (phosphate buffered saline containing 0.15 M NaCI, 1.86 mM KH,PO,, 4.8 mM K,HPO,, pH 7.4) for at least 2 hat room temperature. Stock solutions of valinomycin and nigericin were solubilized in absolute ethanol then diluted in PBS to a final concentration of ethanol of less than 0.5% v/v. 2,5Diphenyloxazole (PPO), scintillation grade, was obtained from Koch Light Ltd, U.K. RPM1 1640 media (with glutamine, without NaHCO,) and Foetal Calf Serum (FCS) were purchased from Cytosystems P/L (Castle Hill, N.S.W., Australia). All other chemicals were reagent grade commercial products. Deionized water was used in all preparative procedures. Cell cultures. Crithidia luciliae was obtained from Dr S. Whittingham of the Waiter and Eliza Hall Institute, Melbourne, Australia and were grown aerobically in RPM1 1640 medium plus 10% FCS and I % L-glutamine at pH 7.4 in a 26’C incubator. Cultures were inoculated in 1 1glass culture flasks by inoculating 500 ml medium with 50 ml of stock stationary phase cells (2 x 10’ ml ‘). Cells were harvested when they reached a density of 7.6 x 10” ml ’ at approximately 72 h after seeding. Cell cultures were harvested by centrifugation at 2500 g for 15 min at 26°C. The pelleted cells were washed twice and then resuspended in PBS immediately prior to assay to a density of4.7 x 10” ml ‘. The concentration of all Crithidia suspensions was enumerated using a haemocytometer with Improved Neubauer ruling observed under phase contrast microscopy. Human erythrocyres. Whole blood containing human type 0 erythrocytes was collected from normal healthy volunteers, provided by the New South Wales Red Cross Blood Transfusion Service and washed in PBS. After centrifugation at 3000 g for 5 min the supernatant and white cell layer were removed by aspiration. The cells were washed a further two times in PBS, with removal of any remaining white cells between washings, and finally resuspended in PBS. Erythrocytes were used within 7 days of collection. Nucleoside transport measurements. Nucleoside transport over short time intervals was measured by a modified version of the established procedure reported previously (Gero,

Scott, O’Sullivan & Christopherson, 1989). To initiate the experiment, cells (4.7 x 10’ ml ‘) in 100 ~1 PBS were added to 1.5 ml Eppendorf centrifuge tubes containing 150 ~1 phthalate oil mix (density 1.03 g ml-‘) with 100 ~11 of radiolabelled permeant layered on top of the oil. Transport intervals (2-30 s at 26°C) were ended by centrifugation at 16,000 g for 20 s which pelleted cells under the oil layer. Thus the radioactivity incorporated into the cell pellet below the oil represented only the fraction transported across the membranes of intact C. luciliae. Background radioactivity due to the extracellular volume of permeant present in the cell pellet was measured by control experiments containing [UY]inulin in the medium in place of the nucleoside permeant. [‘4C]lnulin cannot permeate the cells and so determines the extracellular volume of the pellet. [IH]H,O WiS used to determine total cell water space; it distributes evenly throughout the pellet, is able to penetrate the cells and therefore represents the total volume occupied by the cell pellet and any liquid trapped. The contribution of radioactivity from the extracellular space in the pellet of C. luciliae was determined for each experiment and was shown to be approximately 31% under these experimental conditions. The cell pellet was processed as described by Knodler, Schofield &Edwards (1992) and counted in a Packard Model 300C liquid scintillation spectrometer. For inhibition studies, the compound to be tested, diluted in PBS, was incorporated into the assay by mixing with the radiolabelled permeant prior to the addition of the cells. Kinetic assays were carried out as described above using a variation of substrate concentration. The kinetic constants of K,, and V,,,, were determined by computer analysis using a program to fit the data to MichaelissMenten kinetics (Cleland, 1979). RESULTS Transport

of physiological

nucleosides

The transport of various physiological nucleosides across the plasma membrane of C. luciliae was studied over periods up to 30 s by a rapid sampling technique using centrifugation through inert oil to separate the radioactive permeant from the cells. C. luciliae transported the purine nucleosides, adenosine, deoxyadenosine, guanosine, deoyxguanosine and to a lesser extent, the pyrimidine nucleoside, thymidine. The transport ofeach nucleoside was linear for at least 10 s. A comparison of the rates for several nucleosides is shown in Fig. 1. Adenosine and guanosine were transported across the plasma membrane of the cells at comparable rates (0.3 pmol ~1 cell water-’ SK’, at least two times more rapidly than thymidine (0.17 pmol ~1 cell water-’ s-l). Uridine and deoxycytidine were not transported into C. luciliae to any significant extent. Although not shown, deoxyadenosine was transported across the plasma membrane at a similar rate to adenosine and guanosine whereas deoxyuridine was not transported. A comparison of the influx of physiological adenosine, D-adenosine, with the non-physiological isomer, L-adenosine, into C. luciliae demonstrated that

Adenosine

10

10

0

transport

in Crithidia luciliae

40

30

TIME (s) 0.5

0

FIG. 1. Transport

of radiolabelled nucleosides into C. luciliae. The transport of 1 PM concentrations of [‘Hladenosine (0), [‘Hlguanosine (0). [‘Hlthymidine (Cl), [3H]deoxycytidine (A) and [3H]uridine (a) into C. luciliae was measured after exposure of the cells to the permeant for short intervals from 2-30 sat 26°C. The reaction was stopped by centrifugation of the cells through an inert oil layer as described in Materials and Methods. The radioactive content of the cell pellet was expressed as pmol permeant transported per ~1 cell water after subtraction of background attributable to the extracellular water space in the centrifuged cell pellet. Values depicted represent mean values of triplicate experiments.

J

jj = z 3

4

,

0

5

(

,

10

15

[Adenosinel

1.5

1

(pM)-1

,t5[ ,~~,~ oualwsine o&MM)

-0.5

1

0,”

Adenosinel

,

,

I

I

20

25

30

35

pM

0

0.5

1

Guanosine-

(FM)-]

1.5

2

FIG. 3. Lineweaver-Burk analysis of the transport of (a) adenosine and (b) guanosine into C. luciliae. (a) rH]Adenosine transport was determined in the absence (0) and presence of 10 PM deoxyadenosine (dAdo) (0) or 10 FM inosine (Ino) (A). (b) The insert represents the relationship between velocity and substrate concentration ranging from 0 to 12.5 pM. Data represent average values of triplicate 10 s timepoints.

FIG.

2. Transport of L-adenosine into C. luciliae. The transport of increasing concentrations of [‘H]L-adenosine at 10 s timepoints is depicted. These results represent mean values of at least three experiments. L-adenosine could enter the cell but at a much lower rate. Kinetic analysis at increasing concentrations of L-adenosine indicated that this compound entered the cell by simple diffusion with a rate of 0.025 pmol ~1 cell water-’ PM-' s-’ rather than by facilitated transport (Fig. 2). Kinetic

studies

kinetically characterize the nucleoside transporter activity, the apparent affinities of D-adenosine To

and guanosine for the transporter was determined. Apparent K,,, values were 9.34 f 1.30 PM with a maximum velocity of 2.3 & 0.25 pmol ~1 cell water-’ s-’ for adenosine (Fig. 3a) and 10.6 f 2.60 FM with a maximum velocity of 1.40 & 0.28 pmol ~1 cell water-’ s-’ for guanosine (Fig. 3b). Adenosine transport was inhibited competitively by deoxyadenosine with a K, value of 18.0 pM, whilst inosine had no effect (Fig. 3a). In order to determine the number of nucleoside transporters which may function independently in C. luciliae, competition experiments were performed between adenosine, guanosine, inosine and other nucleosides. As illustrated in Fig. 4, adenosine and

1042

S.T. HALL~~u~.

%TRANSPORT

COMPETITOR

SUBSTRATE

FIG. 4. Effects of competing nucleosides on the transport of radiolabelled adenosine, guanosine, and inosine. The effects of 1 pM concentrations of adenosine (Ado), tubercidin (Tub), guanosine (Guo), and inosine (Ino) upon the translocation of 10 pM [‘Hladenosine (Ado), [“Hlguanosine (Guo) or [Vlinosine (Ino) across the membrane of C. luciliae are depicted. All values expressed as a percentage of the control where the control represents 100% transport in the absence of the competing nucleoside (hatch, n ). Hatch ( ) represents inhibitors of the adenosine transporter whilst (0) represents inhibitors of the guanosine transporter. The data represent mean values of triplicate 5 s timepoints.

tubercidin at 100 pM, inhibited the transport of 1 pM radiolabelled adenosine, whilst 100 pM guanosine or inosine had no effect. Conversely, the transport of either radiolabelled 1 ,UM guanosine or inosine was inhibited by 100 PM cold guanosine or inosine, whilst 100 pM adenosine or tubercidin had no inhibitory effect. In addition deoxyadenosine and sangivamycin were shown to compete with the adenosine transporter, whilst deoxyguanosine competed with the guanosine/inosine transporter (data not shown). The results of the kinetic analysis and the competition assays demonstrated that C. luciliae contained two nucleoside transporters, each with separate substrate specificities which were mutually exclusive to each other. Inhibitor studies on nucleoside transport in C. luciliae Nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole are potent inhibitors of nucleoside transport in mammalian cells (Paterson & Cass, 1986). The effects of these compounds on the transport of adenosine (1 pM) and inosine (6 PM) into C. luciliae is shown in Table 1. All cells were exposed to the radioactive pet-meant and the test compound simultaneously. Neither 10 PM NBMPR, 5 pM dilazep nor 5 PM dipyridamole significantly inhibited the ability of C. luciliae to transport either adenosine or inosine, in stark contrast to the inhibitory effect of these compounds against both adenosine and inosine

transport in human erythrocytes. The latter have been used as a model for the mammalian nucleoside transport system. In order to further characterize the mechanism of nucleoside transport in C. luciliae, the effect of various inhibitors, ionophores and sulphydryl agents, known to interfere with other eukaryotic transport systems were tested against the transport of adenosine (1 pM) into C. luciliae. The cells were preincubated.with the compounds for 5 min prior to each transport assay. None of sodium cyanide (1 mM), ouabain (1 ITIM), valinomycin (10 PM), nigericin (0.3 mM), iodoacetate N-ethylmaleimide (1 mM) or a(1 rnM), aminooxyacetic acid (1 ItIM)had any significant effect in reducing the transport of adenosine into C. luciliae. However, both the sulphydryl reactive agents, p-HMB and p-CMBS, showed significant inhibition of adenosine transport. ID, determination for p-CMBS for both adenosine and guanosine gave values of 15 and 52 ,u~,respectively. After each transport assay the cells were evaluated microscopically for cell motility. All cells showed normal viability in the presence of the inhibitor demonstrating that the inhibition was not due to cell death. DISCUSSION The Trypanosomatidae family and all parasites studied to date are unable to synthesize purines de novo and are therefore forced to salvage these essential nitrogenous compounds from the host. Results from these investigations have shown the existence of two kinetically distinguishable nucleoside transporters in C. Zuciliaewith high affinities for their purine nucleoside substrates. The apparent Km values for the adenosine transporter of 9.3 f 1.30 PM and for the guanosine transporter of 10.6 f 2.60 PM are approximately 5-fold lower than that obtained for mammalian cells (Paterson & Cass, 1986). Thus, the parasite is able to effectively salvage purine nucleosides from its host when the purines are present at low concentrations. Similar results have been demonstrated for L. donovani promastigotes (Aronow et al., 1987) and for T. cruzi (Finley et al., 1988) indicating that there may be a common system for nucleoside transport in the Trypanosomatidae family of protozoa. The importance of the transport of purine nucleosides in C. Zuciliae is exemplified by the inability of Crithidia to transport purine bases into the cell. Kidder, Dewey & Nolan (1978) have demonstrated that under normal culture conditions purine bases were not transported into C. fasciculata and that the transport of purine bases only occurred in cells grown in media deplete of any other purines. The importance of the transport of nucleosides in

Adenosine TABLE

I-EFFECT

OF MAMMALIAN

in Crithidialuciliae

transport

1043

NUCLEOSIDE TRANSPORT INHIBITORSAGAINST THE TRANSPORT OF NUCLEOSIDESIN c. b4Sik’

% Inhibition C. luciliae

Compound Adenosine NBMPR (10 ,UM) Dilazep (5 FM) Dipyridamole (5 PM)

Human

Inosine

Adenosine

Erythrocytes Inosine

12.0+6.5@=3)

5.4Zt9.1

83.3 f 3.6

88.1 f 3.1

5.9 f 0.8 0

0 6.8f2.6

67.1 f 6.4 64.7f 1.5

79.0f 1.8 78.3* 1.3

Crithidia is further illustrated by the discovery of 3’and S-nucleotidases which are located at the cell surface of C. luciliae. Thus, prior to being taken up by Crithidia, extracellular purines, either in the form of nucleotides or nucleic acids, are hydrolysed by the nucleotidases to form nucleosides which are then available for transport (Gottlieb, 1985; 1989; Gottlieb, Mackow & Neubert, 1988). It has been known for some time that certain trypanosomatids can be grown in culture in the presence of only one purine source such as adenosine (Marr et al., 1978). Consequently, all purines must be fully interconvertable, although they are not equally effective at stimulating growth. In L. donovani, for instance, adenosine is the purine of preference for growth, followed by guanosine. Inosine could only support growth after a lag period of two days (Marr et al., 1978). In C. luciliae, adenosine and guanosine were transported at the greatest rate (Fig. 1), followed by inosine (data not shown). The results are similar to those reported for the transport into L. donovani promastigotes (Aronow et al., 1987), in that adenosine and inosine were rapidly transported. However, C. luciliae appeared not to transport uridine, in contrast to L. donovani promastigotes, probably due to the presence of an active de now pyrimidine pathway in C. luciliae (Tampitag & O’Sullivan, 1986). The kinetic and competition data support the existence of two distinct nucleoside transport systems in C. luciliae with separate substrate specificities. Of the nucleosides tested, adenosine, deoxyadenosine, the adenosine analogues, tubercidin and sangivamycin, and the pyrimidine nucleoside thymidine were transported by the first nucleoside transport system. Furthermore, the inhibition of adenosine by deoxyadenosine was competitive substantiating a common site of transport. The second site was specific for guanosine, deoxyguanosine and inosine. The nonphysiological adenosine isomer, L-adenosine, was not transported by either transporter but diffused across the cell membrane at a constant non-saturable rate.

Two nucleoside permeation sites have also been reported for the related trypanosomids L. dunovani (Aronow et al., 1987) and for T. cruzi (Finley et al., 1988). However, the specificities of the substrates for each transporter vary slightly in each organism. L. donovani promastigotes transported adenosine, uridine, formycin B and pyrimidine nucleosides via the first transporter and guanosine and inosine via the second. For T. cruzi, the first was specific for the purine nucleosides, whilst the second was specific for pyrimidine nucleosides and tubercidin. A comparison of the nucleoside transport process of C. lucikae with mammalian nucleoside transport systems reveals several major differences. Firstly, in mammalian cells, nucleosides are transported into the cell by a transport protein in the plasma membrane (for reviews see Paterson & Cass, 1986; Jarvis, 1987) which has a broad substrate specificity, accepting both ribosides and deoxyribosides of both purine and pyrimidine nucleosides as well as synthetic nucleosides of a remarkable structural diversity (Plagemann & Wohlhueter, 1980; Paterson, Kolassa & Cass, 1981), whilst C. luciliae, as well as L. donovani and T. cruzi, each possess two independent transporters with nonoverlapping substrate specificity. Secondly, NBMPR, dilazep and dipyridamole, potent inhibitors of the mammalian nucleoside transporter (Paterson & Cass, 1986) were ineffective against either of the nucleoside transport processes in C. luciliae. The results herein indicate significant differences between parasitic and host nucleoside transport. As purine salvage is essential for the survival of parasitic protozoa, the high affinity of the two transporters for nucleosides in C. luciliae are an effective adaptation of the parasites to be able to successfully compete with the host to meet their own purine metabolic requirements. As C. iuciliae, and the procyclic forms of T. cruzi and L. donovani all occupy the same environment in the insect host, it is of interest that in each case, the parasite has an advantage over the host organism for obtaining its purine requirements.

S. T. HAI _L et al.

1044

These results may also have important chemotherapeutic implications for the treatment of trypanosomal diseases. Due to the high afinity of the C. luciliae

nucleoside transporter for adenosine and guanosine, any cytotoxic analogue of these compounds such as tubercidin, would be incorporated into the parasite more rapidly than into mammalian cells. In addition, there is the possibility of the development of a chemotherapeutic regime of simultaneous administration of two compounds, in which the first, a toxic nucleoside, destroys the viability of the parasite, whilst a second compound, a mammalian nucleoside transport inhibitor such as NBMPR, protects the mammalian cells from the toxicity of the first compound by blocking its transport into the mammalian host cells. Acknowledgements-The authors wish to thank Prof. P. J. Schofield for his interest and suggestions during this project and Prof. W. J. O’Sullivan for his comments on the manuscript. This work was supported by the National Health and Medical Research Council of Australia and the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. REFERENCES ARONOW B., KAUR K., MCCAR~AN K. & ULLMAN B. 1987. Two high affinity nucleoside transporters in Leishmania donovani. Molecular and Biochemical Parasitology 22: 2937. CLELAND W. W. 1979. Statistical analysis of enzyme kinetic data. Methods in Enzymology 63: 103-138. DAVEY R. A., MAYRHOFERG. & EY P. L. 1992. Identification of a broad specificity nucleoside transporter with affinity for the sugar moiety in Giardia intestinalis trophozoites. Biochimica et Biophysics Acta 1109:172-178. FINLEY R. W., COONEY D. A. & DVORAK J. A. 1988. Nucleoside uptake in Trypanosoma cruzi: analysis of a mutant resistant to tubercidin. Molecular and Biochemical Parasitology 31: 133-140. GATI W. P.. STOYKE A. S. W., GERO A. M. & PATERSONA. R. P. 1987. NBMPR insensitive nucleoside permeation in mouse erythrocytes infected with Plasmodium yoelii. Biochemical and Biophysical Research Communications 145: 1134-1141. GERO A. M., BUGLEDICHE. M. A., PATERSONA. R. P. & JAMIESONG. P. 1988. Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes. Molecular and Biochemical Parasitology 27: 159-l 70. GERO A. M. 1989. Alteration in nucleoside permeability in Plasmodium falciparum and Babesia bovis infected erythrocytes. Molecular and Biochemical Parasitology 35: 269-276.

GERO A. M., SCOTT H. V., O’SULLIVAN W. J. & CHRISTOPHERSONR. 1. 1989. Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites. Molecular and Biochemical Parasitology 34: 87-98. GERO A. M. & UPSTON J. M. 1992. Altered membrane permeability: a new approach to malaria chemotherapy. Parasitology Today 8: 283-286. GOTTLIEBM. 1985. Enzyme regulation in a trypanosomatid: Effect of purine starvation on levels of 3’ nucleotidase activity. Science 221:72-74. GOTTLIEB M., MACKOW M. C. & NEWBERT T. A. 1988. Crifhidia luciliae: Factors affecting the expression of 3’nucleotidase/nuclease activity. Experimental Parasitology 66: 108-117. GO~TLIEB M. 1989. The surface membrane 3’-nucleotidase/ nuclease of Trypanosomatid Protozoa. Parasitology Today 5: 257-260. HAMMOND D. J. & GUTTERIDGE W. E. 1984. Purine and pyrimidine metabolism in the Trypanosomatidae. Molecular and Biochemical Parasitology 13: 243-261. HARRIS D. I., BEECHEY R. B., LINS~EAD D. & BARREI-TD. 1988. Nucleoside uptake by Trichomonas vagina/is. Molecular and Biochemical Parasitology 29: 105-l 16. JARVIS S. M. 1987. Kinetic and molecular properties of nucleoside transporters in animal cells. In: Topics & Perspectives in Adenosine Research (Edited by GERLACH E. & BECKERB. F.), pp. 102-l 17. Springer, Berlin. KIDDER G. W., DEWEYV. C. &NOLAN L. L. 1978. Transport and accumulation of purine bases by Crithidia fasciculata. Cell Physiology 96: 165-170. KNODLER L. A., SCHOFIELDP. J. & EDWARDSM. R. 1992. Glucose transport in Crithidia luciliae. Molecular and Biochemical Parasitology 56: 1-14. MARR J. J., BERENS R. L. & NELSON D. J. 1978. Purine metabolism in Leishmania donovani and Leishmania braziliensis. Biochimica et Biophysics Acta 544: 36&371. PATERSONA. R. P., KOLASSAN. & CASS C. E. 1981. Transport of nucleoside drugs in animal cells. Pharmacological Therapy 12: 515-536. PATERSON A. R. P. & CASS C. E. 1986. Transport of nucleoside drugs in animal cells. In: Membrane Transport of Antineoplastic Agents, Vol. 118 (Edited by GOLDMAN 1. D.), pp. 309-329. Pergamon Press, Oxford. PLACEMANN P. W. G. & WOHLHUETER R. M. 1980. Permeation of nucleosides and nucleic acid bases and nucleotides in animals cells. Currenr Topics in Membrane Transport 14: 225-330. SHERMAN I. W. 1984. Metabolism. In: Antimalarial Drugs, Handbook of Experimental Pharmacology (Edited by PETERS W. & RICHARDS W. H. G.), pp. 31-81. Springer, Berlin. TAMPITAG S. SC O’SULLIVAN W. J. 1986. Enzymes of pyrimidine biosynthesis in Crithidia luciliae. Molecular and Biochemical Parasitology 19: 125-134.