Separation of endocytic vesicles in Nycodenz gradients |
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Authors: | G M Kindberg T Ford R Blomhoff D Rickwood T Berg |
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Institution: | 1. Institute for Nutrition Research, University of Oslo, P.O. Box 1046, Blindern, 0316 Oslo 3, Norway;1. Department of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, United Kingdom |
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Abstract: | 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. |
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