Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Abstract

Galactosylated BSA (gaIBSA) and its conjugate to horseradish peroxidase (gaIBSA-HRP) enter the galactose-specific pathway of hepatocytes . 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 x 106 g~min (LP fraction) and have an equilibrium density of 1 .11-1 .13 g/ml in sucrose gradients (Quintart, J., P. J . Courtoy, J . N . Limet, and P. Baudhuin, 1983, Eur . J. Biochem ., 131 :105-112) .Such low density fractions, prepared from rats given gaIBSA-HRP, were incubated for 30 min at 25°C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H202 in buffered sucrose . Upon equilibration in a second sucrose gradient, the gaIBSA-HRP distribution shifted towards higher 01 .19 g/ml) density, but the bulk of protein remained at low density . In the absence of H202, gaIBSA-HRP distribution was also found at low density . As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of gaIBSA-HRP-containing organelles after incubation with DAB and H zOz is attributed to the trapping of HRP-oxidized DAB inside the host organelles .If the low density fractions isolated from a rat injected with [3 H]gaIBSA-HRP were mixed in vitro with similar fractions from another rat given [' 4C]gaIBSA, the 3 H distribution shifted after DAB cytochemistry, but the '4C distribution was essentially unaffected . By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed .In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles . The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes .After receptor-mediated internalization (3, 28), ligands are rapidly transferred to an "intermediate compartment" (34,37), often referred to as "endosomes" (14) or "receptosomes" (37). To assess the purity of subcellular fractions enriched in ligand-containing organelles, we recently used ligands conjugated to horseradish peroxidase (HRP),' an approach that proved successful in intact liver (29,33). Glutaraldehydefixed fractions were incubated in 3,3'-diaminobenzidine ' Abbreviations used in this paper: DAB, 3,3'-diaminobenzidine; gaIBSA, galactosylated BSA; gaIBSA-HRP, gaIBSA conjugated to horseradish peroxidase (HRP) .(DAB) and HZOz (12) and processed for electron microscopy . The organelles containing ligand-HRP were identified by the DAB reaction product (23). We report here that the DAB procedure can also be applied to unfixed subcellular fractions. Moreover, the accumulation of polymerized DAB inside HRP-containing organelles induces a major increase in their equilibrium density, while other organelles of the same fraction are essentially unaftècted.It has already been reported that cytochemical procedures may be applied to the isolation of specific organelles with endogeno...

Degradation of Mutant Influenza Virus Hemagglutinins Is Influenced by Cytoplasmic Sequences Independent of Internalization Signals

“…Like the proteins that appear to have distinct degradation sequences, cross-linked proteins are degraded with half-times that are severalfold longer than the period required to transport HRP (42,51,59) or viruses (44,60) to lysosomes. The degradation rates reported for these proteins are an order of magnitude slower than their internalization rates.…”

“…To determine if the HAϩ8 present in less dense gradient fractions actually resided inside early endosomes, and not in membranes co-migrating with them, cells expressing HAϩ8 were allowed to internalize HRP for 5 min before they were chilled and a post-nuclear membrane preparation was prepared. One half of this preparation was reacted with diaminobenzidine and H 2 O 2 , which results in the deposition of a dense reaction product inside any vesicles containing HRP (51,52). This reaction product has two effects.…”

Prolonged Antigen Persistence Within Nonterminal Late Endocytic Compartments of Antigen-Specific B Lymphocytes

“…The cells were then washed to remove unbound HEL and incubated at 37°C for the time (in hours) indicated in the upper right corner of each panel to allow internalization of HEL-BCR complexes. The B cells were then fixed, permeabilized, and stained for Ag (using the 2D1 murine IgG1 anti-HEL mAb followed by anti-muIgG1-btn and streptavidin-Texas Red) and either DM (A, C, E, G, and I) or DO (B, D, F, H, and J) using rabbit anti-DM or HRP-labeled Ag and took advantage of the observation that delivery of HRP-labeled ligands to various endocytic compartments allows the HRP-catalyzed DAB-mediated cross-linking of the constituent proteins of those vesicles (23)(24)(25). The results presented in Fig.…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…13,19 However, the similarity in the densities of endocytic vesicles, Golgi membranes, and other smooth membranes has led to the development of methods for specifically modifying the density of endocytic vesicles based on their ligand content. 21,27 One of these procedures relies on the shift density that occurs after loading the endocytic structures with lipoproteins (␤-very low density lipoprotein or LDL) in the hepatic cell. 27 Thus, the separation of endosomes from other organelles is based on the low density of the lipoprotein-filled endosomal vesicles.…”

“…A number of methods have been developed for the isolation of hepatic endosomes. 13,[19][20][21][22][23][24][25][26][27] In general, subcellular fractionation explores the differences in the equilibrium densities between organelles or the electric charges of membrane fractions (free-flow electrophoresis). 13,19 However, the similarity in the densities of endocytic vesicles, Golgi membranes, and other smooth membranes has led to the development of methods for specifically modifying the density of endocytic vesicles based on their ligand content.…”