title: Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.
abstract: 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.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Canada.
authors.forename: J R
authors.initials: JR
authors.lastname: Hammond
authors.forename: R M
authors.initials: RM
authors.lastname: Johnstone
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004176:Dipyridamole / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D051379:Mice; D013868:Thioinosine / Q000493:pharmacokinetics / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014529:Uridine / Q000493:pharmacokinetics*
chemical_list: D005960:Glucosides; C018619:octyl-beta-D-glucoside; D013868:Thioinosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine; D000241:Adenosine; D014529:Uridine