ISOLATION OF HEAVY ENDOSOMES FROM DOG PROXIMAL TUBULES IN SUSPENSION

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endo cytosis leading to the appearance of FITC-albumin into intracellular v esicular structures. The isolation of brush border membrane vesicles ( BBMV) from the dog kidney proximal tubules in suspension by the magnes ium precipitation technique leads to the copurification of a large pop ulation of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable i n endosomes and BBM vesicles. However, the acridine orange acidificati on assay showed a V-type ATPase-dependent acidification in endosomes b ut not in BBMV, demonstrating a different orientation of the proton pu mps in these structures. SDS-PAGE analysis also showed significant dif ferences in protein pattern of vesicles and endosomes. The most notabl e difference was the presence of 42-44 kDa and 20-24 kDa proteins in B BMV and their complete absence in endosomes. Western blot analysis ide ntified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different d istribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endo somes. The morphological aspect (electron microscopy) and sedimentatio n of endosomes in a 50% Percoll gradient identified these structures a s ''heavy endosomes'' (buoyant density D=1.036 g/ml). Flow cytometry a nalysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of th ese intracellular organelles. Western blot analysis using anti-FITC an d anti-collagenase antibodies allowed quantification of the FITC-album in and collagenase A in the purified endosomes. Our results indicate t hat heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probabl y by soluble proteins. The suspension of tubules thus offers a experim ental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.