Abstract
An osmotic lysis technique was developed to induce transient permeability in human placental microvillous membrane vesicles. The degree of vesicle opening and resealing was quantitated using the fluorescent markers, 6-carboxyfluorescein and fluorescein dextran. Compared to freeze-thaw and sonication methods, hypotonic lysis was significantly more efficient, causing 90% lysis with 90% subsequent resealing under optimal conditions. The transient increase in vesicle permeability permitted the unrestricted entry of macromolecules with molecular masses up to 70,000 kDa. Passive transport of water, protons, and erythritol and carrier-mediated transport of l-valine and sodium-proton exchange were unaltered by the lysis/resealing procedure. Bovine tracheal vesicles were lysed to an extent similar to placental microvillous vesicles, but rabbit renal cortical brush border and basolateral membranes were lysed to a lesser extent (∼60%). These results show that hypotonic lysis is a suitable method for the loading and trapping of macromolecules in isolated membrane vesicles for studies of intracellular regulation of transport.
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