Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry.
The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D013861:Thiocyanates; D012710:Serum Albumin, Bovine; D008985:Monensin; D016650:Fluorescein-5-isothiocyanate