title: Isolation of highly purified, functional endosomes from toad urinary bladder.
abstract: Endosomes are difficult to isolate as they share size and density properties with much more abundant cellular organelles such as mitochondria. In cultured cell lines the tandem use of charge-dependent isolation techniques and differential centrifugation is necessary to isolate endosomes. Endosomal populations of the toad urinary bladder are of special interest because they are thought to contain a water channel. Understanding of the molecular structure of the water channel has been constrained, as there is currently no practical method to isolate functional water-channel-containing vesicles. This study reports the tandem use of charge-dependent techniques and centrifugation to isolate populations of endosomes from the toad urinary bladder. To purify water-channel-containing vesicles aqueous two-phase partition was utilized to fractionate a preparation partially purified by differential centrifugation. Populations of endosomes were analysed by small-particle flow cytometry techniques. A 5-fold enrichment in endosomes, achieved with aqueous two-phase partition, allowed us to identify two populations of endosomes of diverse size in a toad bladder endosomal fraction. Preenrichment also improved the efficiency of flow cytometry sorting, allowing isolation of the two endosomal populations in sufficient quantities for secondary analysis. A population of larger endosomes had very high water permeability, indicating the presence of water channels. The two populations had different SDS/PAGE fingerprints. Electron micrographs of the flow-sorted material shows a uniform population of membrane vesicles devoid of mitochondria and other identifiable cellular organelles. Hence, aqueous two-phase partition and flow cytometry allow identification of two populations of endosomes in the toad urinary bladder which have diverse structural and functional properties. Isolation of functional water-channel-containing vesicles allows co-localization of water-channel function with candidate water-channel proteins.
authors.affiliation: University of Wisconsin Hospitals and Clinics, Madison.
authors.forename: T G
authors.initials: TG
authors.lastname: Hammond
authors.forename: D J
authors.initials: DJ
authors.lastname: Morré
authors.forename: H W
authors.initials: HW
authors.lastname: Harris
authors.forename: M L
authors.initials: ML
authors.lastname: Zeidel
mesh_terms: D000818:Animals; D002024:Bufo marinus; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D008854:Microscopy, Electron; D015388:Organelles / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D009995:Osmosis; D011506:Proteins / Q000032:analysis; D001743:Urinary Bladder / Q000648:ultrastructure*
chemical_list: D011506:Proteins