Abstract
We have developed a novel method for isolating specific endocytic vesicles using magnetic ligands and high-gradient magnetic separation. Ligands were prepared by coating extremely fine ferrite particles (10-20nm) with bovine serum albumin and then conjugating asialoglycopeptides. These ligands were introduced into rat liver by perfusion at 16 or 37°C, or by injection through the tail vein. The ligand particles were observed as electron-dense small grains in membrane-bound vesicles in Kupffer as well as parenchymal cells by electron microscopy. Livers were taken out, homogenized and lightly centrifuged. The supernatant was pumped into a separator glass tube filled with very fine ferritic stainless steel fibers and placed in a magnetic field of 0.9-2T. Vesicles containing ferrite particles were collected with a high efficiency (ca. 70% of endocytosed magnetic ligands). About 70% of uptake appeared to be mediated by the asialoglycoprotein receptors. The captured vesicles were practically free from marker enzymes for plasma membranes, endoplasmic reticulum, and Golgi apparatus. Lysosomal enzyme activity of the vesicles increased with the time of perfusion at 37°C but not at 16°C. Protein composition of the captured vesicles was analyzed by one- and two-dimensional gel electrophoresis. The composition changed characteristically with time on perfusion at 16 and 37°C. The present method provides a powerful tool to collect prelysosomal endocytic vesicles containing specific ligands and lysosomes fused with these specific endocytic vesicles.
