Isolation and characterization of apical membrane vesicles of the rat distal colon

In this study a method for the isolation of apical membrane vesicles of the rat distal colon was developed. It is based on the purification of intact membrane caps followed by separation of the vesiculated apical membranes on a discontinous sucrose gradient. Purification of the apical membrane vesicles revealed an 11-fold enrichment of the marker enzyme alkaline phosphatase compared with the homogenate, while marker enzymes of other subcellular structures showed negligible enrichments and recovery of activity. The membrane fluidity (lipid structural order) of the isolated membranes measured from the fluorescence anisotropy by several fluorophores also coincided with the typical structural order of apical membranes of the rat colon. Transport studies with the fluorescent dye acridine orange implied that a diffusion potential independent, amiloride-sensitive Na+ -H+ exchange mechanism is present in the isolated apical membranes. Furthermore, the results suggest that a possible short chain fatty acid (SCFA) absorption by simple passive diffusion of the undissociated form, preceded by intraluminal protonation of the SCFA anion, is not provided by this Na+ -H+ exchange transport in the luminal membrane of the absorptive cell.