Nucleoside transport in human erythrocytes. Apparent molecular weight of the nitrobenzylthioinosine-binding complex estimated by radiation-inactivation analysis.
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ABSTRACT:
SUBMITTER: Jarvis SM
PROVIDER: S-EPMC1162102 | biostudies-other | 1980 Aug
REPOSITORIES: biostudies-other
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Project description:Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 X 10(-20) mol/cell per s at 25 degrees C compared with 254 X 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).
| S-EPMC1162103 | biostudies-other
Project description:A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.
| S-EPMC1153932 | biostudies-other
Project description: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.
| S-EPMC1152683 | biostudies-other
Project description:Nitrobenzylthioinosine, a potent nucleoside-transport inhibitor, binds to high-affinity sites on the human erythrocyte membrane. This binding is a specific interaction with functional nucleoside-transport sites. The protein(s) responsible for high-affinity nitrobenzylthioinosine binding was purified 13-fold by treatment of haemoglobin-free 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with Triton X-100 and passage of the soluble extract through a DEAE-cellulose column equilibrated with Triton X-100. Void-volume fractions were collected and treated with Bio-Beads SM-2 to remove detergent. These fractions contained 31% of the starting nitrobenzylthioinosine-binding activity. They also contained D-glucose-sensitive cytochalasin B-binding activity. Nitrobenzylthioinosine binding to the partially purified preparation was saturable (apparent Kd 1.6 nM) and inhibited by nitrobenzylthioguanosine, dipyridamole and uridine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pooled void-volume fractions revealed the presence of only two detectable protein bands, the broad zone 4.5 (containing glucose-transport protein) and a small amount of band 7.
| S-EPMC1162748 | biostudies-other
Project description:Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.
| S-EPMC1163535 | biostudies-other
Project description:We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.
| S-EPMC1131985 | biostudies-other
Project description:The DosiVox programme is used to reconstruct radiation transport in complex depositional environments. Using two archaeological case studies from ancient Egypt, the burial environments for a selection of ceramic vessels are reconstructed using the DosiVox programme, allowing the simulation of the emission and transport of radiation throughout these burial environments. From this simulation we can extract the external dose rate of the archaeological samples, a measurement necessary for determine a luminescence age. We describe in detail how DosiVox can be used to best advantage at sites with complex depositional histories and highlight that DosiVox is a valuable tool in luminescence dating. This work illustrates that DosiVox is, at present, unparalleled in reconstructing a more accurate and detailed external gamma dose rate which can significantly improve upon simplistic scaled geometric models.
| S-EPMC6960934 | biostudies-literature
Project description:The kinetics of [3H]nitrobenzylthioinosine binding to human erythrocyte membranes was studied. The pseudo-first-order association was linear and consistent with a simple bimolecular reaction mechanism between nitrobenzylthioinosine and the nucleoside-transport mechanism. Dissociation of the [3H]nitrobenzylthioinosine complex at 22 degrees C was also linear (apparent k-1 congruent to 0.20 min-1). Adenosine was a competitive inhibitor of equilibrium high-affinity [3H]nitrobenzylthioinosine-binding activity (apparent Ki 0.1 mM). Dissociation of the [3H]nitrobenzylthioinosine-membrane complex was faster in the presence of adenosine and uridine, and this effect was proportional to the nucleoside concentration. Nucleoside concentrations less than 1 mM had no significant effect on the dissociation rate constant. In contrast, dissociation was slower in the presence of high concentrations (micromolar) of dipyridamole. Low concentrations of dipyridamole (2-200 nM) and nitrobenzylthioinosine concentrations as high as 2.5 microM had no effect on the rate of [3H]nitrobenzylthioinosine dissociation. These results are discussed in terms of possible distinct inhibitor and permeation sites, and are suggested to be consistent with both a single-site model for the binding of nitrobenzylthioinosine and permeant to the same site, or an allosteric-site model in which permeant and inhibitor bind to different sites.
| S-EPMC1152559 | biostudies-other
Project description:The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.
| S-EPMC1152939 | biostudies-other
Project description: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.
| S-EPMC1152505 | biostudies-other