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S M Jarvis

Publications and source records attributed to S M Jarvis.

At least 73 records · Page 4Linked to original sources
PubMed1987

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.

Animals↗
PubMed1986

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.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the human erythrocyte glucose transporter with 8-azidoadenosine.

8-Azidoadenosine was employed as a possible covalent probe of the erythrocyte nucleoside transporter. 8-Azidoadenosine was shown to enter human erythrocytes by a saturable mechanism (apparent Km for influx 80 microM) that was inhibited by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, and competitively inhibit uridine influx and NBMPR binding. Irradiation with UV light of human erythrocyte membranes or a partially purified preparation of the nucleoside transporter in the presence of [3H]8-azidoadenosine and dithiothreitol (as a free radical scavenger) resulted in selective covalent incorporation into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr 66,000-45,000). Covalent labeling of band 4.5 was inhibited by adenosine, uridine, and inosine, but NBMPR had no effect. Surprisingly, D-glucose and cytochalasin B, but not L-glucose and cytochalasin E, blocked covalent attachment of the ligand. No incorporation of radioactivity into membranes from rabbit and pig erythrocytes was observed, cells which transport nucleosides rapidly, but have little or no functional glucose carrier. Limited treatment with trypsin of unsealed human erythrocyte membranes photolabeled with [3H]8-azidoadenosine yielded a single radioactive fragment of Mr 19,000, a pattern identical to that obtained with [3H]cytochalasin B-labeled membranes. These results suggest that, despite 8-azidoadenosine being a permeant for the nucleoside transporter, under photoactivation 8-azidoadenosine preferentially labeled the glucose carrier.

Adenosine↗
PubMed1986

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Adenosine↗
PubMed1986

Radiation inactivation of the human erythrocyte nucleoside and glucose transporters.

The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.

Blood Proteins↗
PubMed1986

Trans-stimulation and trans-inhibition of uridine efflux from human erythrocytes by permeant nucleosides.

The ability of nucleoside permeants to accelerate the efflux of uridine from human erythrocytes has been compared. In contrast to uridine, 2-chloroadenosine acted as a trans-inhibitor of uridine efflux from fresh human erythrocytes, and adenosine had little effect. These results are consistent with the lower maximum velocity for influx of 2-chloroadenosine and adenosine as compared with uridine and demonstrate that trans acceleration experiments do not discriminate between transported and non-transported permeants for the human erythrocyte nucleoside carrier.

2-Chloroadenosine↗
PubMed1986

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate.

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 microM) 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 microM. 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 (apparent Km for uridine 50 +/- 18 and 163 +/- 28 microM, respectively). The two transport systems also differed in their sensitivity to pCMBS. NBMPR-insensitive uridine transport was inhibited by pCMBS with an IC50 of approximately 25 microM, while 1 mM pCMBS 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 beta-mercaptoethanol, suggesting that the pCMBS-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 (apparent Kd 46 +/- 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 degrees 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 Mr 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 and pCMBS.

4-Chloromercuribenzenesulfonate↗
PubMed1986

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes.

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.

Affinity Labels↗
PubMed1986

Nitrobenzylthioinosine: an in vivo inhibitor of pig erythrocyte energy metabolism.

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.

Adenosine Diphosphate↗
PubMed1986

Nitrobenzylthioinosine-sensitive nucleoside transport system: mechanism of inhibition by dipyridamole.

Dipyridamole-mediated inhibition of nucleoside transport by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated diffusion system in mammalian erythrocytes was investigated. [3H]Dipyridamole was a competitive inhibitor of uridine equilibrium exchange influx into guinea pig erythrocytes (apparent Ki 1 nM). Analysis of the results using total inhibitor levels instead of cell-free inhibitor concentrations increased the apparent Ki value to 7 nM. Similarly, [3H]dipyridamole inhibition of zero-trans-[14C] uridine influx was consistent with simple competitive inhibition (apparent Ki 1.4 +/- 0.7 nM). In contrast, [3H]dipyridamole behaved as a noncompetitive inhibitor of zero-trans-[14C]uridine efflux (apparent Ki 0.7 +/- 0.2 nM). In a second series of experiments, [3H]dipyridamole was found to bind to a single class of high affinity sites on plasma membranes from human erythrocytes (apparent Kd 0.65 +/- 0.07 nM) with a maximum number of binding sites similar to that determined with the nucleoside transport inhibitor NBMPR. Binding of dipyridamole to these sites was blocked by the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine, and dilazep and in a competitive manner by adenosine and uridine (apparent inhibition constants 0.1 and 0.9 mM, respectively). These inhibition constants are similar to the apparent Km for adenosine and uridine equilibrium exchange in human erythrocytes. These results are consistent with the notion that, in mammalian erythrocytes, dipyridamole interacts with the NBMPR-sensitive transporter at the same site as NBMPR, which is preferentially located on the outer surface of the cell membrane totally or partially within the permeation site.

Animals↗
PubMed1985

2-Chloroadenosine, a permeant for the nucleoside transporter.

Human erythrocytes were shown to possess a saturable uptake mechanism for 2-chloroadenosine (apparent Km 23 microM, 22 degrees). Uptake by this route was inhibited by nitrobenzylthioinosine, uridine and adenosine, but adenine had no effect. In addition, uridine caused the countertransport of 2-chloroadenosine and vice versa. 2-Chloroadenosine was also shown to be an apparent competitive inhibitor of uridine influx (apparent Ki value of 33 microM) and high-affinity nitrobenzylthioinosine binding (apparent Ki 0.18 mM). The apparent Ki value for inhibition of uridine influx was close to the apparent Km value for 2-chloroadenosine uptake. Previous studies [Jarvis et al., Biochem. J. 208, 83 (1982)] have demonstrated that dog erythrocytes do not possess a saturable transport system for uridine and adenosine. Similarly, in the present study, the entry of 2-chloroadenosine into dog erythrocytes was slow and linear with concentration. Nitrobenzylthioinosine (NBMPR) had no effect on the uptake of 2-chloroadenosine into dog erythrocytes. These results demonstrate that 2-chloroadenosine enters human erythrocytes by the same nucleoside carrier as other nucleosides. It is suggested from these data that the previous explanation that the inability of nucleoside transport inhibitors to potentiate the pharmacological effects of 2-chloroadenosine was due to the failure of the nucleoside carrier to accept 2-chloroadenosine as a permeant may have to be reassessed.

2-Chloroadenosine↗
PubMed1985

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.

Affinity Labels↗
PubMed1985

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.

Biological Transport, Active↗
PubMed1985

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.

Adenosine↗
PubMed1984

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 Mr of 45000-65000.

Affinity Labels↗
PubMed1984

Identification of the nucleoside transporter in cultured mouse lymphoma cells. Photoaffinity labeling of plasma membrane-enriched fractions from nucleoside transport-competent (S49) and nucleoside transport-deficient (AE1) cells with [3H]nitrobenzylthioinosine.

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.

Adenosine↗
PubMed1984

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 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Biological Transport↗