Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes.
The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.
Kwong
F Y
FY
Department of Biochemistry, Faculty of Medicine, Chinese University of Hong Kong, Sha Tin.
Tse
C M
CM
Jarvis
S M
SM
Choy
M Y
MY
Young
J D
JD
D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D005960:Glucosides; D006151:Guanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D033703:Nucleoside Transport Proteins; D010777:Photochemistry; D012995:Solubility; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D013872:Thionucleosides / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D005960:Glucosides; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013872:Thionucleosides; D006151:Guanosine; C002121:6-(4-nitrobenzylthio)guanosine; C018619:octyl-beta-D-glucoside; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine