Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules.
The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein 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 extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.
D000818:Animals; D004705:Endocytosis; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008175:Lung Neoplasms / Q000503:physiopathology*; D051379:Mice; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate