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.
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.
Agbanyo
F R
FR
McEachern Laboratory, University of Alberta, Edmonton, Canada.
Vijayalakshmi
D
D
Craik
J D
JD
Gati
W P
WP
McAdam
D P
DP
Asakura
J
J
Robins
M J
MJ
Paterson
A R
AR
Cass
C E
CE
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005960:Glucosides; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D033703:Nucleoside Transport Proteins; D009705:Nucleosides / Q000378:metabolism*; D012995:Solubility; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D013872:Thionucleosides / Q000737:chemistry*
D000345:Affinity Labels; D002352:Carrier Proteins; D005960:Glucosides; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D009705:Nucleosides; D013872:Thionucleosides; C066257:5'-S-(2-aminoethyl)-N(6)-(4-nitrobenzyl)-5'-thioadenosine; C018619:octyl-beta-D-glucoside; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine