Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography.
The nucleoside transporter has been purified by passage of a preparation of human erythrocyte-membrane band-4.5 proteins through a column of immobilized antibodies specific for the glucose transporter. This procedure removed greater than 99.8% of the glucose transporters and achieved an approx. 18-fold purification of the nucleoside transporter, constituting a 478-fold purification from erythrocyte membranes. The isolated protein migrated as a single broad band of average apparent Mr 55,000 on SDS/polyacrylamide gels and bound approx. 0.6 mol of nitrobenzylthioinosine/mol of polypeptide, with a Kd of 1.1 +/- 0.14 (S.E.M.) nM. Upon reconstitution into large unilamellar phospholipid vesicles it catalysed the uptake of uridine with an apparent specific activity 6-fold greater than that of the unfractionated band-4.5 proteins. Furthermore, the purified nucleoside transporter was not labelled on Western blots by monoclonal antibody raised against the glucose transporter. It is concluded that the nucleoside transporter has been purified to near homogeneity.
Kwong
F Y
FY
Department of Biochemistry & Chemistry, Royal Free Hospital School of Medicine, University of London, U.K.
Davies
A
A
Tse
C M
CM
Young
J D
JD
Henderson
P J
PJ
Baldwin
S A
SA
D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D033703:Nucleoside Transport Proteins; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D010446:Peptide Fragments; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D005947:Glucose; D014529:Uridine