Separation of endocytic vesicles in Nycodenz gradients

✍ Scribed by Grete M. Kindberg; Terence Ford; Rune Blomhoff; David Rickwood; Trond Berg

• Book ID: 102987153
• Publisher: Elsevier Science
• Year: 1984
• Tongue: English
• Weight: 647 KB
• Volume: 142
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(84)90489-5

✦ Synopsis

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.