The cellular uptake of horseradish peroxidase and its poly(lysine) conjugate by cultured fibroblasts is qualitatively similar despite a 900-fold difference in rate.
When horseradish peroxidase (HRP) is conjugated to poly(L-lysine) of molecular weight (MW) 13,000, its transport into cultured L929 fibroblasts in 1 hour at 37 degrees C is increased 918-fold. The kinetics of uptake are linear with time and concentration, reflecting a process of nonreceptor-mediated adsorptive endocytosis. Neither HRP-poly(Lys) conjugate nor free poly(Lys) of any size cause any increase in fluid phase endocytosis. All evidence indicates that the covalently bound poly(Lys) increases the binding of HRP to the cell surface and that this adequately accounts for an increased internalization occurring at a steady rate. In the absence of Ca++, both surface binding and uptake of the conjugate, but not of free HRP, are decreased. Cytochalasin B does not significantly inhibit the transport of either form of HRP. The half-lives of HRP and HRP-poly(Lys) are 7.6 and 5.6 hours, respectively, when measured over a period of 12 hours. Electron microscopic analysis of cells that have ingested comparable amounts of HRP shows that the intracellular localization of free and conjugated HRP are comparable and that both are found inside the same array of structures. Thus HRP, which binds very poorly to the cell surface and is considered a fluid-phase marker for the "contents" of pinocytotic vesicles, and HRP-poly(Lys), which binds very strongly to the cell surface and is considered a membrane marker for adsorptive endocytosis, are taken up along the same endocytotic pathway. Moreover, despite the fact that neither markers are transported by receptor-mediated endocytosis, both are seen in structures that were described as receptosomes. It appears, therefore, that the pathways utilized in receptor-mediated transport are available to all other forms of pinocytosis.
D000818:Animals; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000502:physiology; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007526:Isoelectric Point; D007700:Kinetics; D051379:Mice; D010544:Peroxidases / Q000378:metabolism*; D011107:Polylysine; D013329:Structure-Activity Relationship
D011107:Polylysine; D000001:Calcimycin; D003571:Cytochalasin B; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002118:Calcium