Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate.

摘要: The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with aid an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated a newly developed cleavable reagent, bismaleimidoethoxy propane, following thiolation both proteins. Noncleavable conjugates also prepared. At saturating conjugate concentrations, uptake rate for averaged over first 2 h is about 6.5 fmol/million cells/min. Incubation loaded in fresh medium 30 min analysis cell pellets supernatants reveal that 1) previously cell-associated label, only intact (about 50% label) returned medium; 2) most remaining material chromatographically coincident free Fab some contribution from fragments. In contrast, noncleavable contained transferrin-F(ab'), conjugates. These results are consistent receptor-mediated followed by hydrolysis acidified endosomes resulting concentration within prelysosomal intracellular compartment. A protein shuttle such as may therefore be ketal based cross-linkers load foreign molecules addition, these data provide independent confirmation low pH compartment cycle. This new methodology applicable other cases receptor/ligand trafficking report compartments morphological analysis. Since receptors overexpressed tumors, antineoplastic agents could targeted tumors system might particularly useful combatting tumor export antitumor occurring multidrug resistance.