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Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

1987; Elsevier BV; Volume: 904; Issue: 1 Linguagem: Inglês

10.1016/0005-2736(87)90091-5

1879-2642

FRANCIS Y. P. KWONG, Chung‐Ming Tse, Simon M. Jarvis, Matthew Y.M. Choy, James D. Young,

Drug Transport and Resistance Mechanisms

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.