Ligand-specific isolation of endosomes and lysosomes using superparamagnetic colloidal iron dextran glycoconjugates and high gradient magnetic affinity chromatography.
We have developed a ligand-specific method for the visualization, isolation, and biochemical characterization of cell surface and intracellular membranes mediating endocytic transport. Iron dextran particles (FeDex) bearing either covalently conjugated galactosyl bovine serum albumin (GalBSA/FeDex) or asialofetuin (ASF/FeDex) are bound by the asialoglycoprotein receptor (ASGP-R) of HepG2 cells and transported to lysosomes with kinetics indistinguishable from those of free GalBSA or ASF. FeDex particles, which have a 3 to 5 nm electron-dense colloidal iron core, can be visualized by electron microscopy. Following incubation of GalBSA/FeDex with HepG2 cells at 37 degrees C, FeDex particles are seen at the cell surface, in endosomes, and in lysosomes. Surface membrane and intracellular organelles bearing a sufficient number of FeDex particles can be efficiently isolated from disrupted cells by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes and endosomal/lysosomal membranes isolated by HIMAC are 35 to 40-fold enriched for GalBSA/FeDex or ASF/FeDex relative to the postnuclear supernatant. Alkaline phosphodiesterase I (APDE) and galactosyltransferase are each enriched 8-fold in the plasma membrane fraction prepared by HIMAC whereas neither beta-galactosidase nor glucose-6-phosphatase are detected in this fraction. The intracellular membrane fraction, containing both endosomes and lysosomes, is enriched for galactosyltransferase and beta-galactosidase but not for APDE or glucose-6-phosphatase. Use of FeDex conjugates in conjunction with HIMAC provides an effective method for ligand-specific isolation of membranes and correlation of morphological and biochemical characteristics.
Becich
M J
MJ
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Baenziger
J U
JU
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D011992:Endosomes / Q000737:chemistry* / Q000648:ultrastructure; D005696:Galactosidases / Q000032:analysis; D005700:Galactosyltransferases / Q000032:analysis; D005952:Glucose-6-Phosphatase / Q000032:analysis; D006001:Glycoconjugates; D006801:Humans; D007505:Iron-Dextran Complex; D008024:Ligands; D008247:Lysosomes / Q000737:chemistry* / Q000648:ultrastructure; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D012710:Serum Albumin, Bovine
D006001:Glycoconjugates; D008024:Ligands; D012710:Serum Albumin, Bovine; D007505:Iron-Dextran Complex; D005700:Galactosyltransferases; D005952:Glucose-6-Phosphatase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D005696:Galactosidases