Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of 3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for 3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of 3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with 3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where 3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte 3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte 3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside 1.0% (w/v) + asolectin] solubilized over 90% of the 3H]NBMPR-binding sites, with near-complete retention of 3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating 3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.