Purine Nucleobase Transport Research Articles

Monitoring HSVtk suicide gene therapy: the role of [18F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [18F]FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [18F]FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [18F]FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour-bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2-chloroadenosine did not significantly affect the [18F]FHPG uptake in vitro. Thymidine and uridine significantly decreased [18F]FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [18F]FHPG uptake in C6tk and C6 tumours decreased from 3.0±0.5 to 1.0±0.2 after infusion of adenine. Thus, in our tumour model, [18F]FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier.

A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle.

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of ∼1 μM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major.

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K m =0.91±0.19 μM ) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K m values of 105 and 134 μM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine ( K m values 0.28–1.5 μM) as well as cytidine ( K i =32 μM ). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3′ and 5′ hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably π–π-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.

Six Related Nucleoside/Nucleobase Transporters fromTrypanosoma brucei Exhibit Distinct Biochemical Functions

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na +-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63±3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood–brain barrier, while the free diffusion is negligible. The derived value of K m (20.72±2.42 μM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61±2.28 μM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

Drug uptake via nutrient transporters in Trypanosoma brucei.

Drug uptake via nutrient transporters in Trypanosoma brucei.

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family.

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.

Transport of purine nucleobases by COS-1 cells occurs via a nitrobenzylthioinosine-insensitive (ei) nucleoside transporter

Transport of purine nucleobases by COS-1 cells occurs via a nitrobenzylthioinosine-insensitive (ei) nucleoside transporter

Purine nucleobase transport in bloodstream forms of Trypanosoma brucei brucei is mediated by two novel transporters

The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 μM) by guanosine was biphasic; ≈90% of transport activity was inhibited with a K i value of 10.8±1.8 μM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their K m values (123±15 nM and 4.7±0.9 μM for H2 and H3, respectively) although the V max values (1.1±0.2 and 1.1±0.1 pmol (10 7 cells) −1 s −1, n=3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H +-ATPase inhibitors, N, N′-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [ 3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H +/hypoxanthine symporter.

Hypoxanthine uptake through a purine-selective nucleobase transporter in Trypanosoma brucei brucei procyclic cells is driven by protonmotive force.

The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22 degrees C was rapid and saturable, exhibiting an apparent Km of 9.3 +/- 2.0 microM and a Vmax of 4.5 +/- 0.8 pmol x (10(7) cells)(-1) x s(-1). All the natural purine nucleobases tested (Ki 1.8-7.2 microM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi), the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH 7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electrogenic. These data show that hypoxanthine uptake in T. b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.

Purine nucleobase transport in bloodstream forms of Trypanosoma brucei brucei.

brucei [2]. However, almost nothing is currently known about nucleobase uptake by the long- slender forms of this species that exist in the mammalian bloodstream, though these bloodstream forms are know to possess two distinct transporters for adenosine, one of which displays high affinity for adenine [3]. In this report, we describe the characterisation of purine nucleobase transport in

Inhibition of purine nucleobase transport in human erythrocytes and cell lines by papaverine: Investigation of structure-activity relationship

Papaverine was found to be an effective inhibitor of hypoxanthine transport not only in human erythrocytes, but also in the human cell lines HL60 (myeloic) and U937 (monocytic). ic 50 values for inhibition of hypoxanthine influx ranged from 6 to 20 μM. In erythrocytes papaverine was found to be a non-competitive inhibitor of hypoxanthine equilibrium-exchange transport with a K i value of ≈13 μM, which is in close agreement with the respective ic 50 values estimated for zero-trans influx of hypoxanthine. In addition papaverine also had a slight inhibitory effect on unmediated nucleobase transport, most likely due to a perturbation of the membrane lipid environment. Several papaverine analogs were tested for their inhibitory effect on nucleobase transport. Only ethaverine was as effective as papaverine. Drotaverine and berberine were moderately inhibitory while laudanosine had no inhibitory effect at all. Isoquinoline acted as a very weak inhibitor.

Purine nucleoside and nucleobase cell membrane transport in Giardia lamblia.

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines. This study investigated purine nucleoside and nucleobase transport utilizing rapid uptake determinations. Nucleoside substrate/velocity curves exhibited the hyperbolic kinetics of a saturable carrier-mediated system. Deoxynucleosides exhibited a much lower affinity for the transporter. Inhibition studies confirmed the relative carrier affinities of these ribonucleosides and deoxyribonucleosides. The nucleobase adenine did not exhibit saturation kinetics at a comparable substrate range, and did not inhibit nucleoside transport. Dipyridamole markedly inhibited nucleoside but not nucleobase transport, confirming the separate entry pathways. When cells were depleted of ATP, the velocity of nucleoside and nucleobase transport was unchanged, indicating that it is a non-energy-dependent process. Three nucleoside analogs, formycin A, adenine arabinoside and 7-deazaadenosine, were studied. Transport kinetics ranged widely among this group and could not completely account for their cytotoxic effect. When the apparent Km and Vmax of the nucleosides were compared, an approximately linear relationship (r2 = 0.95) was noted. This suggests that a high affinity of the nucleoside permease for the substrate retards disassociation of the substrate-carrier complex, slowing net influx.

Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel “inhibitor-stop” assay.

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter.

A novel type of somatic mutation that causes the expression of a high-affinity purine base permease (B. Aronow, D. Toll, J. Patrick, P. Hollingsworth, K. McCartan, and B. Ullmann, Mol. Cell Biol. 6:2957-2962, 1986) has been inserted into nucleoside transport-deficient S49 cells. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possessed an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possessed an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells could transport nucleosides, suggesting that the expression of the high-affinity base transporter did not reverse the mutation in the nucleoside transport system. The transport of purine bases by both AE1HADPAB2 and AE1HADPAB5 cells was much less sensitive than that by wild-type cells to inhibition by dipyridamole, 4-nitrobenzylthionosine, and N-ethylmaleimide, potent inhibitors of nucleoside and nucleobase transport in wild-type S49 cells. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild-type cells indicated that the expression of the high-affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high-affinity purine base transporter of mutant cells and the nucleoside transport function of wild-type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild-type cells.

Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter.

A novel type of somatic mutation that causes the expression of a high-affinity purine base permease (B. Aronow, D. Toll, J. Patrick, P. Hollingsworth, K. McCartan, and B. Ullmann, Mol. Cell Biol. 6:2957-2962, 1986) has been inserted into nucleoside transport-deficient S49 cells. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possessed an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possessed an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells could transport nucleosides, suggesting that the expression of the high-affinity base transporter did not reverse the mutation in the nucleoside transport system. The transport of purine bases by both AE1HADPAB2 and AE1HADPAB5 cells was much less sensitive than that by wild-type cells to inhibition by dipyridamole, 4-nitrobenzylthionosine, and N-ethylmaleimide, potent inhibitors of nucleoside and nucleobase transport in wild-type S49 cells. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild-type cells indicated that the expression of the high-affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high-affinity purine base transporter of mutant cells and the nucleoside transport function of wild-type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild-type cells.

Expression of a novel high-affinity purine nucleobase transport function in mutant mammalian T lymphoblasts.

The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019, 1986). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 microM DPA and 11 microM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 microM hypoxanthine-11 microM azaserine-10 microM DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 microM concentrations of hypoxanthine, guanine, and adenine 15- to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.