Separation of endocytic vesicles in Nycodenz gradients.
The endocytosis of 125I-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after 1/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligand-containing endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies.
D000643:Ammonium Chloride; D000818:Animals; D001212:Asialoglycoproteins; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D004705:Endocytosis; D060748:Fetuins; D007472:Iohexol; D008024:Ligands; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014283:Triiodobenzoic Acids; D000509:alpha-Fetoproteins / Q000378:metabolism*
D001212:Asialoglycoproteins; D060748:Fetuins; D008024:Ligands; D014283:Triiodobenzoic Acids; D000509:alpha-Fetoproteins; C017029:asialofetuin; D000643:Ammonium Chloride; D007472:Iohexol