Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.     

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.     

Design, synthesis, and evaluation of 5'-S-aminoethyl-N(6)- azidobenzyl-5'-thioadenosine biotin conjugate: a bifunctional photoaffinity probe for the es nucleoside transporter

A bifunctional biotinylated photoaffinity label for the nitrobenzylmercaptopurine riboside (NBMPR)-sensitive (es) nucleoside transporter (ENT1) has been synthesized and evaluated. This new probe,5'-S-aminoethyladenosine-N(6)-azidobenzyl-5'-thioadenosine biotin conjugate (SAEATA-14-biotin), exhibited high-affinity binding to the es transporter in K562 cells as determined by flow cytometry, with a K(i) of 2.69 nM in competition against 5-(SAENTA)-x8-fluorescein. It also exhibited covalent linking to the es transporter in BeWo cell membranes upon UV irradiation. This new bifunctional probe is a potential tool for determining the amino acid residues involved in ligand binding at the NBMPR-binding site of the ENT1 nucleoside transporter, as well as for the purification of the transporter.     

Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.     

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.     

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.     

Identification of the erythrocyte nucleoside transporter as a band 4.5 polypeptide. Photoaffinity labeling studies using nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR) was employed as a covalent probe of the erythrocyte nucleoside transporter. This nucleoside analogue, a potent inhibitor of nucleoside transport, binds tightly (KD = 10(-10) - 10(-9) M) but reversibly to specific sites on the carrier mechanism. High intensity UV irradiation of intact human erythrocytes, isolated "ghosts," and "protein-depleted" membranes in the presence of [3H]NBMPR and dithiothreitol (as a free radical scavenger) under nonequilibrium and equilibrium binding conditions resulted in selective covalent incorporation of 3H into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr = 45,000-65,000). Covalent labeling of band 4.5 protein(s) under equilibrium binding conditions was inhibited by nitrobenzylthioguanosine, dipyridamole, uridine, and adenosine. A similar photolabeling pattern was observed using membranes from pig erythrocytes. In contrast, no incorporation of radioactivity into band 4.5 was observed under equilibrium binding conditions with membranes from nucleoside-impermeable sheep erythrocytes. These experiments suggest that the human and pig erythrocyte nucleoside transporters are band 4.5 polypeptides, a conclusion supported by previous isolation studies based on the assay of reversible [3H]NBMPR binding activity.     

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.     

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.     

[3H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.     

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.     

Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.     

Mammalian nitrobenzylthioinosine-sensitive nucleoside transport proteins. Immunological evidence that transporters differing in size and inhibitor specificity share sequence homology.

Polyclonal antibodies were raised against the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter of human erythrocyte membranes. On Western blots of these membranes they labeled the broad "band 4.5" region (average apparent M(r) 55,000), which contains both the nucleoside and glucose transport proteins. However, they did not recognize the glucose transporter when this was prepared free of nucleoside transporter by expression from a cDNA clone. Their specificity for the nucleoside transporter was confirmed by the ability to immunoadsorb NBMPR- but not cytochalasin B-binding sites from a detergent-solubilized mixture of band 4.5 proteins. Although a large proportion of the antibodies recognized extracellular epitopes, these appeared to be located primarily on the polypeptide moiety of the glycoprotein, as demonstrated by the ability of the antibodies strongly to label the deglycosylated transporter (apparent M(r) 45,000) on Western blots. The antibodies were species-cross-reactive, recognizing nucleoside transporters from pig and rabbit erythrocytes and from rat liver. The pig protein is similar to the human transporter in its inhibitor sensitivity but is considerably larger (apparent M(r) 57,000 after deglycosylation). In contrast, the rat protein is similar in size to the human transporter (apparent M(r) 45,000 after deglycosylation) but much less sensitive to the inhibitors dilazep and dipyridamole. These findings indicate that despite their differences in size and inhibitor specificity, the NBMPR-sensitive nucleoside transporters of these mammalian species are related in amino acid sequence.     

Photoaffinity labelling of the nucleoside transporter of cultured mouse lymphoma cells

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [³H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that ³H was present in polypeptides which migrated as a major band with an apparent M_r of 45000–65000.     

Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (less than 50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (greater than 1 microM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (greater than 1 microM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.     

Species Differences in the Binding of [3H]Nitrobenzylthioinosine to the Nucleoside Transport System in Mammalian Central Nervous System Membranes: Evidence for Interconvertible Conformations of the Binding Site/Transporter Complex

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11-4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 less than 20 nM and IC50 greater than 6 microM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitor-induced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.     

The human erythrocyte nucleoside and glucose transporters are both band 4.5 membrane polypeptides.

Human erythrocyte membranes and partially purified nucleoside transporter (band 4.5 and 7) were photoaffinity labelled with 3H-labelled 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine under equilibrium binding conditions. Band 4.5 was the major site of radiolabelling in both preparations. These experiments provide additional evidence to implicate band 4.5 polypeptides in nucleoside permeation, proteins previously shown to be involved in hexose transport.     

Erythrocyte nucleoside and sugar transport. Endo-beta-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins.

Nucleoside- and glucose-transport proteins isolated from human erythrocyte membranes were photoaffinity-labelled with [3H]nitrobenzylthioinosine and [3H]cytochalasin B, respectively, and subjected to endo-beta-galactosidase or endoglycosidase-F digestion. Without enzyme treatment the two radiolabelled transporters migrated on SDS/polyacrylamide gels with the same apparent Mr (average) of 55,000. Apparent Mr (average) values after endo-beta-galactosidase digestion were 47,000 and 48,000 for the nucleoside and glucose transporters respectively, and 44,000 and 45,000 respectively after endoglycosidase-F digestion. In contrast, endo-beta-galactosidase had no effect on the electrophoretic mobility of the nucleoside transporter isolated from pig erythrocytes. This transport system exhibited a higher Mr than the human protein, endoglycosidase-F treatment decreasing its apparent Mr (average) from 64,000 to 57,000. It is concluded that the human and pig erythrocyte nucleoside transporters are glycoproteins containing N-linked oligosaccharide. The data provide evidence of substantial carbohydrate and polypeptide differences between the human and pig erythrocyte nucleoside transporters, but evidence of molecular similarities between the human erythrocyte nucleoside and glucose transporters.     

The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells.

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.