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.     

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.     

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.     

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.     

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.     

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.     

Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells.

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.     

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 rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate

Summary The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 M) by NBMPR was biphasic –35% of the transport activity was inhibited with an IC₅₀ value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 M. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentK _m for uridine 50±18 and 163±28 M, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC₅₀ of 25M, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by -mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [³H]NBMPR binding to the cell membrane (apparentK _d46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [³H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [³H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent M_r on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS.     

A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.     

Water exchange through erythrocyte membranes: p-choloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes . The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37°C. No inhibition was noticed after short incubation at 0°C as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance ( n . m . r . ) method appears as a useful tool for studying changes in water diffusiofl in erythrocyte ghosts with the aim of locating the water channel.     

Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.     

[3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung.

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.     

Water exchange through erythrocyte membranes V. Incubation with papain prevents the p-chloromercuri-benzensulfonate inhibition of water diffusion studied by a nuclear magnetic resonance technique

The changes in water diffusion across human erythrocyte membrane following exposure to proteolytic enzymes and to p-chloromercuribenzene sulfonate (PCMBS) have been studied on isolated erythrocytes suspended in isotonic solutions. Trypsin digested glycophorin without significantly changing the pattern of other polypeptides in erythrocyte membrane. On the contrary, with chymotrypsin or papain an extensive digestion of band 3 protein occured. No changes in water diffusion were noticed after exposure of erythrocytes to trypsin, chymotrypsin or papain. Neither trypsin nor chymotrypsin treatment prevented the inhibition of water diffusion induced by PCMBS. In contrast, exposure of erythrocytes to papain did hamper the inhibitory effect of subsequent incubation with PCMBS. Taking into account the degradation of band 3 protein by papain it appears that the binding site for PCMBS playing a role in the inhibition of water diffusion is located in this protein.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

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.     

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.     

Water permeability in human erythrocytes: identification of membrane proteins involved in water transport

The water permeability of human erythrocytes has been monitored by nuclear magnetic resonance (NMR) before and after treatment of the cells with various sulfhydryl reagents. Preincubation of the cells with N-ethylmaleimide (NEM), a non-inhibitory sulfhydryl reagent, results in a faster and more sensitive inhibition of water exchange by mercurials. The inhibition of water exchange by p-chloromercuribenzene sulfonate (PCMBS) was maximal at a binding of approximately 10 nmol PCMBS per mg protein when non-specific sulfhydryl groups are blocked by NEM. Inhibition by PCMBS has been correlated with the binding of 203Hg to erythrocyte membrane proteins. A significant binding of label to band 3 and the polypeptides in band 4.5 occurs, with approximately 1 mol of mercurial bound per mol of protein. Inhibition of water transport by sulfhydryl reagents does not induce major morphological changes in the cells as assessed by freeze-fracture and scanning electron microscopy.     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport

The reactions of three organic mercurial compounds, chlormerodrin, parachloromercuribenzoate (PCMB), and parachloromercuribenzenesulfonate (PCMBS) with intact red blood cells, hemolyzed red cells, hemoglobin solutions, and hemoglobin-free ghosts have been characterized. Both PCMB and PCMBS react with only 2 to 3 sulfhydryl groups per mole of hemoglobin in solution, whereas chlormerodrin reacts with 6 to 7. In hemoglobin-free ghosts, however, all three reagents react with a similar number of sulfhydryl groups, approximately 4 x 10^-17 moles per cell, or about 25 per cent of the total stromal sulfhydryl groups, which react with inorganic mercuric chloride. In the intact cell the membrane imposes a diffusion barrier; chlormerodrin and PCMB penetrate slowly, whereas PCMBS does not. Kinetic studies of chlormerodrin binding to intact cells reveal that the majority of stromal sulfhydryl groups is located inside the diffusion barrier, with only 1 to 1.5 per cent (or 1 to 1,400,000 sites per cell) located outside of this barrier. Reaction of PCMBS with intact cells is limited to this small fraction on the outer membrane surface. All three reagents are capable of inhibiting glucose transport in the red cell. With chlormerodrin and PCMBS it was demonstrated that the inhibition results from interactions with the sulfhydryl groups located on the outer surface of the membrane.