Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

Kwong, F. Y. P.; Tse, Chung Ming; Jarvis, Simon M.; Choy, M. Y. M.; Young, James D.

doi:10.1016/0005-2736(87)90091-5

Cited by 22 publications

(11 citation statements)

References 49 publications

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“…peptides free of glucose-transporter polypeptides is to use membranes from cells that transport nucleosides but not glucose, such as erythrocytes of adult pigs [19][20][21][22]. Nucleoside-transporter polypeptides of pig erythrocytes have been partially purified from detergent-solubilized protein-depleted membrane preparations by DEAE-cellulose chromatography [23]. Nucleosidetransporter polypeptides enriched in this way were used to raise monoclonal antibodies (mAbs) that recognize NBMPR-binding polypeptides of pig erythrocytes [241.…”

Section: F R Agbanyo and Othersmentioning

confidence: 99%

“…NBMPR and NBTGR were prepared as described previously [26]. [G-3H]NBMPR (23 Ci/mmol), obtained from Moravek Biochemicals, Brea, CA, U.S.A., was purified by using a Spheri-10 RP18 100 mm x 4.6 mm column (Brownlee Labs, Santa Clara, CA, U.S.A.), eluted with a methanol/water gradient. 251I-labelled goat anti-mouse IgG was purchased from du Pont Canada (Mississauga, Ontario, Canada).…”

Section: Experimental Materialsmentioning

confidence: 99%

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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

Agbanyo

Vijayalakshmi

Craik

et al. 1990

Biochemical Journal

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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.

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Section: F R Agbanyo and Othersmentioning

confidence: 99%

Section: Experimental Materialsmentioning

confidence: 99%

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

Agbanyo

Vijayalakshmi

Craik

et al. 1990

Biochemical Journal

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“…Our attempts to inhibit cellular uptake of adenosine by inhibition of the nucleoside transporter with NBMPR [20,42,58,85] or to abrogate stimulation of purinergic receptors utilizing the unspecific purinergic receptor antagonist CGS-15943 [20,42,58,79,85], the adenosine A1-receptor antagonist FSCPX [80], or the adenosine A3 receptor antagonist VUF5574 [61] failed to reverse the inhibitory effect of adenosine on phosphatidylserine exposure. This observation does not rule out, although, that cellular adenosine uptake or activation of receptors by adenosine contributes to or even accounts for the antieryptotic effect of adenosine.…”

Section: Discussionmentioning

confidence: 99%

“…Removal of Cl − leads to exit of KCl and osmotically obliged water and thus to cell shrinkage, a well-known trigger of eryptosis [45]. Adenosine was used at concentrations ranging from 10 to 100 μM, the nucleoside transport inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) [20,42,58,85] …”

Section: Erythrocytes Solutions and Chemicalsmentioning

confidence: 99%

Adenosine protects against suicidal erythrocyte death

Niemoeller

Bentzen

Lang

et al. 2007

Pflugers Arch - Eur J Physiol

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Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The cell membrane scrambling is triggered by an increase in cytosolic Ca(2+) activity and activation of protein kinase C (PKC). Phosphatidylserine exposure fosters adherence of affected erythrocytes to the vascular wall. Thus, microcirculation in ischemic tissues may be impaired by the appearance of eryptotic erythrocytes. Ischemia leads to release of adenosine, which in most tissues leads to vasodilation and protects against cell injury. The present experiments explored whether adenosine influences mechanisms underlying eryptosis. Erythrocyte phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter and cytosolic Ca(2+) activity from Fluo3 fluorescence. Glucose depletion (for 24 or 48 h) significantly increased annexin binding and decreased forward scatter, effects partially reversed by adenosine. The protective effect of adenosine reached statistical significance (s.d.) at> =30 microM. Low Cl(-) solution (Cl(-) exchanged by gluconate for 24 h) similarly increased annexin binding and decreased forward scatter, effects again reversed by adenosine (s.d. at > or =10 and 30 microM, respectively). Similarly, phosphatase inhibitor okadaic acid (OA, 1 microM) and PKC activator phorbol 12-myristate-13-acetate (PMA, 3 microM) significantly enhanced annexin binding and decreased forward scatter. Adenosine significantly blunted the effects of OA and PMA on annexin V binding (s.d. at > or =30 and 10 microM, respectively) and the effect of OA on forward scatter (s.d. at > or =10 microM). In conclusion, adenosine inhibits eryptosis by a mechanism presumably effective downstream of PKC. The effect may participate in the maintenance of microcirculation in ischemic tissue.

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“…Transport inhibition is associated with site-specific high-affinity binding of ligand to the transport mechanism. NBMPR-sensitive nucleoside transporters have been partially purified from both human and pig erythrocytes (-104 and 5 • 103 copies per cell, respectively) and shown to be band 4.5 polypeptides [nomenclature of Steck (1974)] with apparent molecular weights on sodium dodecyl sulfate (SDS)-polyacrylamide gels of 45,000-66,000 (Jarvis and Young, 1981;Kwong et al, 1986Kwong et al, , 1987. A similar nucleoside transporter is present in lower abundance in Nu-permeable sheep red cells (20 sites per cell) but is functionally absent from Nu-impermeable red cells from this species, with respect to both nucleoside transport activity and high-affinity NBMPR binding Young, 1978, 1980).…”

Section: Discussionmentioning

confidence: 99%

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep

Tucker

Young

1988

Biochem Genet

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Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (NuI) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nui). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NPH) is incompletely dominant to that for low activity (NPL). Inheritance data indicate that the Nu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labile NPL gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.

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Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

Cited by 22 publications

References 49 publications

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

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

Adenosine protects against suicidal erythrocyte death

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep

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