scholarly journals 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography

1990 ◽  
Vol 270 (3) ◽  
pp. 605-614 ◽  
Author(s):  
F R Agbanyo ◽  
D Vijayalakshmi ◽  
J D Craik ◽  
W P Gati ◽  
D P McAdam ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Binding Protein ◽  
Broad Band ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Erythrocyte Membranes ◽  
Partial Purification ◽  
Nucleoside Transporter ◽  
Derivatives Of ◽  
Detergent Solutions

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5′-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.

scholarly journals Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

1998 ◽  
Vol 330 (2) ◽  
pp. 997-1001 ◽  
Author(s):  
Antonio FELIPE ◽  
Raquel VALDES ◽  
Belén del SANTO ◽  
Jorge LLOBERAS ◽  
Javier CASADO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Liver Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Plasma Membrane Vesicles

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the ‘epithelial-specific isoform’ called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

Characteristics of membrane transport losses during reticulocyte maturation

1987 ◽  
Vol 65 (10) ◽  
pp. 869-875 ◽  
Author(s):  
Rhoda Blostein ◽  
Eva Grafova
Keyword(s):  
Membrane Transport ◽  
Binding Sites ◽  
Sodium Pump ◽  
Proteolytic Processing ◽  
Atp Depletion ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Losses

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+–glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+–glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

scholarly journals Interferon-γ regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways

2003 ◽  
Vol 375 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Concepció SOLER ◽  
Antonio FELIPE ◽  
José GARCÍA-MANTEIGA ◽  
Maria SERRA ◽  
Elena GUILLÉN-GÓMEZ ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Interferon Γ ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Nucleoside Transporter ◽  
Protein Levels ◽  
Ifn Γ

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-γ (interferon-γ). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-γ was studied using macrophages from STAT1 knockout mice. IFN-γ triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-γ is required for DNA synthesis. Interestingly, IFN-γ led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-γ up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-γ is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-γ in macrophages.

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

1988 ◽  
Vol 66 (8) ◽  
pp. 839-852 ◽  
Author(s):  
James D. Craik ◽  
A. Heather Good ◽  
Russell Gottschalk ◽  
Simon M. Jarvis ◽  
Alan R. P. Paterson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cytochalasin B ◽  
Cultured Cells ◽  
Uv Light ◽  
Binding Protein ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Intact Cells ◽  
Neonatal Pigs ◽  
Limited Digestion

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.

scholarly journals Use of membrane vesicles to estimate the numbers of system y+ and system L amino acid transporters in human erythrocytes

1991 ◽  
Vol 277 (2) ◽  
pp. 565-568 ◽  
Author(s):  
C M Tse ◽  
D A Fincham ◽  
J C Ellory ◽  
J D Young
Keyword(s):  
Amino Acid ◽  
Membrane Vesicles ◽  
Human Erythrocytes ◽  
Transport Systems ◽  
Erythrocyte Membranes ◽  
Amino Acid Transporters ◽  
Nucleoside Transporter ◽  
Uridine Uptake ◽  
System L ◽  
Human Erythrocyte Membranes

We have used equilibrium values for L-leucine and L-lysine uptake by right-side-out vesicles to estimate the membrane abundance (sites/cell) of Na(+)-dependent amino acid transport systems L and y+ in human erythrocytes. All of the intravesicular space was accessible to L-leucine, as judged by comparisons with uridine uptake via the equilibrative nucleoside transporter (10(4) sites/cell). In contrast, only 28% of the total intravesicular space was accessible to L-lysine uptake via system y+. Since human erythrocyte membranes generate an average of approximately 1000 vesicles/cell, these data provide evidence that system L is a relatively high-abundance membrane transport protein in human erythrocytes, while system y+ is present in smaller amounts (approximately 300 copies/cell). Calculated turnover numbers for L-lysine transport by system y+ at 37 degrees C are 24 s-1 for zero-trans influx and 150 s-1 for equilibrium-exchange influx.

scholarly journals Differential uptake of [3H]guanosine by nucleoside transporter subtypes in Ehrlich ascites tumour cells

1992 ◽  
Vol 287 (2) ◽  
pp. 431-436 ◽  
Author(s):  
J R Hammond
Keyword(s):  
Mammalian Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Tumour ◽  
Transport Inhibitors ◽  
Influx Kinetics ◽  
Ascites Tumour Cells ◽  
Ehrlich Ascites Tumour Cells

Intracellular metabolism of [3H]guanosine was minimal (< 15%) during the first 22 s of incubation, and hence reasonable estimates of initial-rate influx kinetics could be derived by using metabolically active cells. Na(+)-dependent concentrative [3H]guanosine uptake was not observed. Data suggest that [3H]guanosine was accumulated primarily via the nitrobenzylthioguanosine (NBTGR)-sensitive subtype of facilitated nucleoside transporter. Incubation of cells with 100 nM-NBTGR significantly decreased the potency of guanosine as an inhibitor of [3H]uridine influx. The Vmax. for [3H]guanosine influx (9.2 pmol/s per microliters) was significantly lower than that for [3H]uridine influx (16 pmol/s per microliters). The Km for transporter-mediated [3H]guanosine influx determined in the presence of 100 nM-NBTGR was 16-fold higher (1780 microM) than that determined in its absence, whereas the Km for [3H]uridine influx was shifted by only 2-fold. In other respects, the cellular accumulations of [3H]guanosine and [3H]uridine were similar; both had Km values of approx. 140 microM for total mediated influx, and both were inhibited similarly by other nucleosides and transport inhibitors. These characteristics, and the fact that guanosine is an endogenous nucleoside, suggest that [3H]guanosine may prove useful as a poorly metabolized, relatively selective, substrate for study of the NBTGR-sensitive nucleoside transport systems of mammalian cells.

scholarly journals Multiple sodium-dependent nucleoside transport systems in bovine renal brush-border membrane vesicles

1991 ◽  
Vol 274 (1) ◽  
pp. 27-33 ◽  
Author(s):  
T C Williams ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Nucleoside Transporter ◽  
Renal Brush Border Membrane ◽  
Renal Brush Border

Na(+)-dependent nucleoside transport was examined in bovine renal brush-border membrane vesicles. Two separate Na+/nucleoside cotransporters were shown to be present: (1) a system specific for purine nucleosides and uridine, designated as the N1 carrier, and (2) an Na(+)-dependent nucleoside transporter that accepts pyrimidine nucleosides, adenosine and analogues of adenosine, designated as the N2 system. Both systems exhibit a high affinity for nucleosides (apparent Km values approximately 10 microM), are insensitive to inhibition by facilitated-diffusion nucleoside transport inhibitors, are rheogenic and exhibit a high specificity for Na+. Na+ increases the affinity of the influx of guanosine and thymidine, nucleosides that serve as model permeants for the N1 and N2 nucleoside transporters respectively. The Na+/nucleoside coupling stoichiometry is consistent with 1:1 for both carriers.

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