Endocytic Pathways

Abstract

Horseradish peroxidase (HRP) can catalyze the hydroxylation of a variety of aromatic compounds, including phenolic substrates such as 3,3’-diaminobenzidine (DAB), as well as tyrosine, phenylalanine, and sialic acid. These latter compounds may partly substitute for DAB as reductant. DAB cytochemistry has been well established for the localization of HRP in fixed tissue (Graham and Karnovsky, 1966). This same principle has been used by Courtoy et al. (1984), to distinquish non-fixed HRP-containing endosomes from cellular organelles of a similar size and equilibrium density. They made use of a conjugate of asialoglycoprotein and HRP to specifically label rat liver endosomes, which are involved in the receptor mediated uptake of asialoglycoproteins, with peroxidase activity. Later, we applied this technique after labeling tissue culture cells with asialoglycoprotein-HRP and transferrin-HRP conjugates, as well as fluid phase-endocytosed HRP (Stoorvogel et al. 1987; 1988; 1989; Geuze et al. 1988). Two major effects of DAB cytochemistry were observed. 1. Intravesicular DAB polymer is formed, and trapped within the vesicle. Due to the high density of the DAB-polymer, HRP containing vesicles are recovered at a much higher equilibrium density in a density gradient following centrifugation than non-HRP-containing microsomes. At increasing DAB concentrations this density shift becomes more pronounced (fig 1). 2. After DAB cytochemistry, proteins present within the HRP-containing compartment can no longer be extracted in a soluble form after lysis of the microsome. Encapsulation of proteins by DAB polymer as well as chemical cross-linking may play a role in this effect.