Part of the
NATO ASI Series
book series (volume 62)
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.
KeywordsHepG2 Cell Sialic Acid Early Endosome Late Endosome Endocytic Pathway
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Ashwell G, and Morell AG (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 41,99–128.PubMedGoogle Scholar
Courtoy PJ, Quintart J, and Baudhuin P (1984). Shift of equilibrium density induced by 3,3’-diaminobenzidi- ne cytochemistry: A new procedure for the analysis and purification of peroxidase-containing organelles. J Cell Biol 98,870–876.PubMedCrossRefGoogle Scholar
Dautry-Varsat A, Ciechanover A, and Lodish HF (1983). pH and the recycling of transferrin receptor during receptor mediated endocytosis. Proc Nad Acad Sci USA 80,2258–2262.CrossRefGoogle Scholar
Geuze HJ, Stoorvogel W, Strous GJ, Slot JW, Bleekemolen JE, and Mellman I (1988). Sorting of mannose 6- phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J Cell Biol 107,2491–2501.PubMedCrossRefGoogle Scholar
Graham RC Jr., and Karnovsky MJ (1966). The early stages of absorbtion of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Hist Chem 14,291–302.CrossRefGoogle Scholar
Klausner RD, Ashwell G, van Renswoude J, Harford JB, and Bridges KR (1983). Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci USA 80,2263–2266.PubMedCrossRefGoogle Scholar
Stoorvogel W, Geuze HJ, and Strous GJ (1987). Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells. J Cell Biol. 104,1261–1268.PubMedCrossRefGoogle Scholar
Stoorvogel W, Geuze HJ, Griffith JM, and Strous GJ (1988). The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J Cell Biol 106,1821–1829.PubMedCrossRefGoogle Scholar
Stoorvogel W, Geuze HJ, Griffith JM, Schwartz AL, and Strous GJ (1989). Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol 108,2137–2148.PubMedCrossRefGoogle Scholar
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