In Amphiuma red blood cells the Na +/H + exchanger is responsible for both intracellular pH and volume regulation. In many other cells, this inducible transporter plays an important role in multiple cellular functions, and its activity can be regulated by a variety of biochemical agents and signals. The presence of intracellular vesicles has been recently reported for this erythrocytes, and a novel hypothesis involving fluid-phase endocytosis as responsible for the regulation of this plasma membrane exchanger has been proposed (Cala et al, unpublished results). In the present study, this hypothesis has been tested, by establishing the presence of several proteins associated with transport vesicles, like caveolae and clathrin coated vesicles. Commercially available antibodies have been employed against caveolin, clathrin, adaptin β and adaptin γ, in order to detect the presence of these proteins in cytosolic, membrane and particulate fraction of Amphiuma erythrocytes. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane by westernblot. The membrane was stained with India Ink, followed by standard Immunoblot with enhance chemiluminescence. The presence of caveolin and adaptin β was detected in membrane and particulate fractions respectively. No adaptin γ, was detected in the erythrocytes by these methods. Taken together, these results indicates that in Amphiuma red blood cells both caveolae and clathrin coated vesicles are present. The potential role of these pathways in the regulation of membrane transporters are currently under investigation.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology