Isolate and Characterise Brush Border Membrane Vesicles and Basolateral Membrane Vesicles from Equine Small Intestine

Membrane vesicles were isolated from the brush border membrane and basolateral membrane domains of equine enterocytes. In order to show that the isolated membrane vesicles derive either from the brush border or basolateral membranes and are pure, they were examined for the enrichment, dysenrichment of abundance, activity of specific marker proteins and enzymes characteristic of the brush border, basolateral and organelle membranes. Villin is a reliable marker of the intestinal brush border membrane. The result showed that there is a single immuno-reactive band for villin at 92.5 kilo Daltons in all 3 regions of the equine small intestine. This band is enriched in membrane vesicles over the homogenates and confirms that the membrane vesicles isolated do originate from the brush border membrane. The enrichment of alkaline phosphatase enzyme specific activity was increased (~ 6 to 11-fold) in BBMV over homogenates, further confirming that the vesicles were of brush border membrane origin. Na+/K+ ATPase is a classical marker protein of the intestinal basolateral membrane. The result showed a typical western blot for the levels of Na+/K+ ATPase in BLMV isolated from the small intestine of horse. The final pellet shows an enrichment of 95 and 40 kDa bands (?- and ?- sub-units of the Na+/K+ ATPase), thus confirming that the vesicles are of basolateral membrane origin. Here the brush border membrane origin of vesicles has been assessed by showing enrichment of villin and alkaline phosphatase. Also the basolateral membrane origin of vesicles has been assessed by determining the enrichment of Na+/K+ ATPase in the basolateral membrane vesicles.