Isolation of highly purified, functional endosomes from toad urinary bladder.
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.
Hammond, TG; Morré, DJ; Harris, HW; Zeidel, ML
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