Nucleoside Transport In Human Erythrocytes Research Articles

Human Erythrocyte Nucleoside Transporter ENT1 Functions at Ice-cold Temperatures

The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed.

Ribavirin uptake by human erythrocytes and the involvement of nitrobenzylthioinosine-sensitive (es)-nucleoside transporters.

1. The major toxicity associated with oral therapy with ribavirin is anaemia, which has been postulated to occur as a result of accumulation of ribavirin triphosphate interfering with erythrocyte respiration. The objective of this study was to determine the mechanism by which ribavirin enters into erythrocytes. 2. Entry into human erythrocytes was examined by measuring influx rates of [3H]-ribavirin alone and with the inhibitor nitrobenzylthioinosine (NBMPR), and by investigating the inhibitory effects of nucleoside and nucleobase permeants on ribavirin transport, by use of inhibitor oil-stop methods. Transport mechanisms were further characterized by assessment of substrates to cause countertransport of ribavirin in preloaded erythrocytes, and by measuring the effects of ribavirin on [3H]-NBMPR binding to erythrocyte membranes. 3. Human erythrocytes had a saturable influx mechanism for ribavirin (Km at 22 degrees C of 440+/-100 microM) which was inhibited by nanomolar concentrations of NBMPR (IC50 0.99+/-0.15 nM). Nucleosides also inhibited the influx of ribavirin (adenosine more effective than uridine) but the nucleobases hypoxanthine and adenine had no effect. In addition, uridine caused the countertransport of ribavirin in human erythrocytes. Entry of ribavirin into horse erythrocytes, a cell type that lacks the NBMPR-sensitive (es) nucleoside transporter, proceeded slowly and via a pathway that was resistant to NBMPR inhibition. Ribavirin was a competitive inhibitor of adenosine influx (mean Ki 0.48+/-0.14 mM) and also inhibited NBMPR binding to erythrocyte membranes (mean Ki 2.2+/-0.39 mM). 4. These data indicate that ribavirin is a transported permeant for the es nucleoside transporter of human erythrocytes. There was no evidence for ribavirin entering cells via a nucleobase transporter.

Immunolocalisation of nucleoside transporters in human placental trophoblast and endothelial cells: evidence for multiple transporter isoforms.

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M(r) 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.

The Inhibition of Nucleoside Transport in Human Erythrocytes by Mioflazine Analogues

Abstract Kinetic analysis of the transport protein (both influx and efflux), usually performed with radiolabelled nucleosides such as adenosine and uridine, has provided a wealth of information regarding the various kinetic and equilibrium parameters (1).

Inhibition of nucleoside transport by nitrobenzylthioformycin analogs

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-((β- d-ribofuranosyl) pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(β- d-ribofuranosyl) pyrazolo[4,3- d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC 50 values for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [ 3H]NBTF yielded a K d of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with K d values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10 −7 to 10 −6 M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides

Time courses of transmembrane equilibration of 2-chloroadenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, cytidine and 2′-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero- trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange ( K ee ) ranged from 22 μM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities ( V ee ) differed in a similar manner, so that the first-order rate constants ( V ee /K ee ) were similar for all nucleosides. The kinetic parameters for 2′-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3′-OH group greatly reduced the affinity of 3t́-deoxyadenosine (cordycepin) for the carrier, 2′,3′-Dideoxynucleosides were transported < 1% as efficiently as 2′- and 3t́-deoxynucleosides. Thus, the 2′- and 3′-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by V ee was 5–10-times greater than that of the empty carrier, whereas the mobility of th adenosine-loaded or 2′-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3′-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as K ee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.

Passive and carrier-mediated permeation of different nucleosides through the reconstituted nucleoside transporter

When reconstituted into proteoliposomes, the human erythrocyte nucleoside transporter catalysed nitrobenzylthioguanosine (NBTGR)-sensitive zero- trans influx of three different nucleosides at broadly similar rates (inosine, uridine > adenosine). However, proteoliposomes also exhibited high rates of NBTGR-insensitive uptake of adenosine, making this nucleoside unsuitable for reconstitution studies. Equivalent high rates of adenosine influx were observed in protein-free liposomes, establishing that this permeability pathway represents simple diffusion of nucleoside across the lipid bilayer. In contrast to adenosine, inosine and uridine exhibited acceptable rates of NBTGR-insensitive uptake. Of the two, inosine is the more attractive permeant for reconstitution experiments, having a 2.5-fold lower basal membrane permeability. Studies of nucleoside transport specificity in reconstituted membrane vesicles should take account of the widely different passive permeabilities of different nucleosides.

Characterization of polyclonal antibodies against the human erythrocyte nucleoside transporter

Characterization of polyclonal antibodies against the human erythrocyte nucleoside transporter

48 PARTIAL PURIFICATION OF THE NUCLEOSIDE TRANSPORTER FROM HUMAN ERYTHROCYTES

The nucleoside transporter of human erythrocytes can be identified by photolabelling with radiolabelled nitrobenzyl-thioinosine, a potent high affinity inhibitor of nucleoside transport in human erythrocytes and some somatic cells. The nucleoside transporter migrates as a broad band on SDS potyacrylamide gels with a molecular weight between 45,000 and 65,000 daltons. This band width probably reflects heterogeneous glycosylation. The nucleoside transporter from human erythrocytes has been partially purified by a combination of high pH washes to remove extrinsic proteins followed by ion exchange chromotography on DEAE-cellulose. In this fashion, the nucleoside transporter has been purified approximately 10 to 20 fold from human erythrocyte ghosts. However, the nucleoside transporter preparation was contaminated by the glucose transporter. Efforts are currently underway to generate proteolytic fragments of the nucleoside transporter that are distinct and separable from the glucose transporter and to separate the glucose transporter from the nucleoside transporter by immunoaffinity chromatographic techniques. The focus of these studies is to generate a sufficiently pure preparation of nucleoside transporter that can be used to obtain amino acid sequence or to generate antibodies that can be subsequently exploited for the isolation of molecular clones encoding the human nucleoside transport system.

Interaction of [3H]dipyridamole with the nucleoside transporters of human erythrocytes and cultured animal cells.

Equilibrium binding of [3H]dipyridamole identified high-affinity (Kd approximately 10 nM) binding sites on human erythrocytes (approximately 5 X 10(5) sites/cell) and on HeLa cells (approximately 5 X 10(6) sites/cell). The equilibration of dipyridamole with these sites on human erythrocytes was compatible with a second-order process which proceeded at 22 degrees C with a rate constant of about 6 X 10(6) M-1 sec-1. Binding of dipyridamole to these sites correlated kinetically with the inhibition of the equilibrium exchange of 500 microM uridine in these cells and was inhibited in a concentration-dependent manner by nucleosides and other inhibitors of nucleoside transport, such as nitrobenzylthioinosine, dilazep and lidoflazine, but not by hypoxanthine, which is not a substrate for the nucleoside transporter of human erythrocytes. The results indicate that the substrate binding site of the transporter is part of the high-affinity dipyridamole binding site. Bound [3H]dipyridamole became displaced from these sites on human erythrocytes by incubation with an excess of unlabeled dipyridamole or high concentrations of nucleosides and inhibitors of nucleoside transport, but neither by hypoxanthine nor sugars. Dissociation of [3H]dipyridamole behaved as a simple first-order process, but the rate constant was about one order of magnitude lower (about 3 X 10(-3) sec-1) than anticipated for typical ligand-protein binding on the basis of the measured association rate and equilibrium constants. The reason for this discrepancy has not been resolved. No high-affinity dipyridamole binding sites were detected on Novikoff rat hepatoma cells, P388, L1210 and S49 mouse leukemia cells or Chinese hamster ovary cells, and their absence correlated with a greater resistance of nucleoside transport in these cells to inhibition by dipyridamole. All cells expressed considerable low affinity (Kd greater than 0.5 microM) and nonspecific binding of dipyridamole.

Proteolytic cleavage of [3H]nitrobenzylthioinosine-labelled nucleoside transporter in human erythrocytes.

The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1 degree C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66 000-45 000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of Mr 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 000-31 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1 degree C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent Mr fragments of 38 000 and 23 000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19 000 which was distinct from the Mr 23 000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments.

Evidence for the asymmetrical binding of [formula omitted] sulphonate to the human erythrocyte nucleoside transporter

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p- chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 μM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.

Adenine nucleotide metabolism and nucleoside transport in human erythrocytes under ATP depletion conditions

The adenine nucleotides of human red cells were labeled by incubation of the cells with [ 3H]adenosine. Then, the cells were incubated in Tris-saline with various supplements that cause the loss of cellular ATP, and the degradation products were quantitated as a function of time of incubation at 37°C. Incubation of the cells with 2.5 or 5 mM iodoacetate, iodoacetamide or 1 mM HCHO in combination with 5 mM KF and 50 mM deoxyglucose, 50 mM d-glucose or 10 mM inosine was most efficient in depleting the cells of ATP (100% in 0.5–1 h) without causing cell lysis. In iodoacetate- and iodoacetamide-treated cells practically all catabolism of ATP occurred via ADP → AMP → IMP → inosine → hypoxanthine with hypoxanthine accumulating in the medium. In HCHO-treated cells and in cells incubated in Tris-saline or in Tris-saline with deoxyglucose with and without KF, a substantial proportion of ATP (up to 50%) was catabolized via ADP → AMP → adenosine → inosine → hypoxanthine . Under all conditions, AMP deamination and IMP and AMP hydrolysis were rate-limiting reactions. IMP degradation was more rapid in iodoacetamide- and HCHO-treated than in iodoacetate-treated red cells. It was also more rapid in fresh than in outdated red cells, and it was inhibited by P i. Treatment with iodoacetamide and HCHO under ATP-depletion conditions resulted in a 60–80% inhibition of uridine transport by the cells. Treatment with iodoacetate or deoxyglucose plus KF had only minor effects on nucleoside transport; thus, cells treated in this manner might be useful for studying the transport of adenosine and deoxyadenosine under conditions were their phosphorylation is prevented.

Reconstitution studies of the human erythrocyte nucleoside transporter.

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.

Identification of the adenosine uptake sites in guinea pig brain.

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.

Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange ( V ee) at 25°C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange ( K ee), and that maximum zero- trans efflux ( V 21 zt) is about 4-times greater than maximum zero- trans influx ( V 12 zt) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25°C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5°C rather than 25°C. At 5°C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero- trans efflux exceeds maximum zero- trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25°C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.

Effect of temperature on kinetics and differential mobility of empty and loaded nucleoside transporter of human erythrocytes.

The transmembrane equilibration of [3H]uridine was measured in human erythrocytes as a function of temperature using rapid kinetic techniques. Arrhenius plots of the maximum velocity of equilibrium exchange were continuous between 5 and 30 degrees C (Ea = 17-20 kcal/mol), but the increase in velocity with increase in temperature leveled off above 30 degrees C. This leveling off did not reflect heat inactivation of the carrier since transport activity was stable for 3 h at 37 degrees C. Transmembrane equilibration of uridine in equilibrium exchange and zero-trans modes at 5, 15, 25, and 35 degrees C conformed to appropriate integrated rate equations derived for the simple transporter. The nucleoside transporter exhibited directional symmetry, but the loaded carrier moved on the average 5 times more rapidly than the empty carrier at 15, 25, and 35 degrees C, but 25-40 times faster at 5 degrees C. This marked shift in differential mobility of loaded and empty carrier between 15 and 5 degrees C was entirely attributable to an impairment of mobility of empty carrier. The Michaelis-Menten constant for equilibrium exchange increased about 3-fold with increase in temperature between 5 and 35 degrees C. The van't Hoff plot of the values was approximately linear and yielded an estimate of the enthalpy of carrier:substrate dissociation of 7.8 kcal/mol.

Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.

Nucleoside transport in human erythrocytes: Nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung

Binding of the potent nucleoside transport inhibitor [ 3H]nitrobenzylthioinosine to rat and guinea pig lung membranes was investigated. Reversible high-affinity binding was found in both species (apparent K D ∼ 0.3nM). Binding was inhibited by nitrobenzylthioguanosine, adenosine and uridine. Dipyridamole was also an effective inhibitor of [ 3H]nitrobenzylthioinosine binding to guinea pig membranes. In contrast, rat membranes were relatively insensitive to dipyridamole. Exposure of site-bound [ 3H]nitrobenzylthioinosine to high intensity U.V. light resulted in the photoaffinity labelling of lung proteins with apparent molecular weights similar to that of the human erythrocyte nucleoside transporter (45,000–65,000).