3/23/2023 0 Comments N linked glycosylationPar photomarquage à des sites spécifiques avec la nitrobenzylthioinosine (NBMPR), nous avons identifié des polypeptides membranaires (masse relative ( M r) 48 000 – 55 000) associés au transport équilibrant des nucléosides dans les cellules leucémiques murines (L1210/C2) cultivées. Key words: nucleoside transport, L1210 cells, nitrobenzylthioinosine, glycoproteins, tunicamycin. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells. Tunicamycin exposures that reduced incorporation of mannose into plasma membrane fractions by > 95% had little, if any, effect on either the affinity ( K d values, 0.1–0.2 nM) or abundance ( B max values, 200 000 – 220 000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 ☌ were altered in a complex way. These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the NBMPR-labeled polypeptides migrated in SDS–polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted NBMPR-labeled polypeptides to the more rapidly migrating form ( M r 41 000 – 48 000). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly ( M r 42 000 – 47 000) during sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Membrane polypeptides (relative mass ( M r) 48 000 – 55 000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with nitrobenzylthioinosine (NBMPR).
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