Linked Life Data

8694908

Source:http://linkedlifedata.com/resource/pubmed/id/8694908

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1996-12-18
pubmed:abstractText
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 endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population 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 in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0022-2631
pubmed:author
pubmed:issnType
Print
pubmed:volume
153
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
59-73
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Isolation of heavy endosomes from dog proximal tubules in suspension.
pubmed:affiliation
Nephrology Laboratory, Centre de Recherche Louis-Charles Simard, Hôpital Notre-Dame de Montréal, Montréal, Québec H2L 4M1, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't