Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique

We have employed a modification of the horseradish peroxidase (HRP)-diaminobenzidine density shift technique of Courtoy et al. (J. Cell Biol., 1984, 98:870-876) to examine the biochemical properties of the endosome. This organelle is involved in receptor recycling and the sorting of internalized receptor ligand complexes. Transferrin covalently bound to HRP was used to place peroxidase activity specifically within the endosome. The peroxidase-catalyzed polymerization of diaminobenzidine within these vesicles causes an increase in buoyant density, thus allowing them to be separated from other membranes. Using this technique we demonstrate that 125I-low density lipoprotein, 131I-epidermal growth factor, and Tf-HRP are internalized into the same endosome. We discovered that the diaminobenzidine reaction product "cross-links" the lumen of the vesicle, rendering vesicular components detergent insoluble. Furthermore, the reaction inactivates enzymatic activities associated with the endosome. Thus, the diaminobenzidine density shift procedure has limited usefulness in studies designed to isolate endosomal constituents. Nonetheless, we have found that the inactivation of enzymatic activities is confined to those endosomes that contain peroxidase. This selectivity allows us to define endosome-specific activities.