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

• Published in: Biochemical journal 1990-09, Vol.270 (3), p.605-614
• Main Authors: AGBANYO, F. R, DAMARAJU VIJAYALAKSHMI, CRAIK, J. D, GATI, W. P, MCADAM, D. P, ASAKURA, J.-I, ROBINS, M. J, PATERSON, A. R. P, CASS, C. E
• Format: Article
• Language: English
• Subjects: Adenosine - analogs & derivatives; Adenosine - chemistry; Affinity Labels; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Biological and medical sciences; Biological Transport; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane - chemistry; Fundamental and applied biological sciences. Psychology; Glucosides; Ligands; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Molecular Weight; Nucleoside Transport Proteins; Nucleosides - metabolism; Proteins; Solubility; Swine; Thioinosine - analogs & derivatives; Thioinosine - metabolism; Thionucleosides - chemistry
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2700605

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.