Simultaneous isolation and characterization of brush border and basolateral membrane vesicles from bovine small intestine.

Abstract

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Alkaline phosphatase and Na/K adenosine triphosphatase served as marker enzymes for the brush border and basolateral membranes, respectively. The brush border and basolateral membrane fractions were enriched 5.1- and 10.1-fold, respectively, over the cellular homogenate. Electron micrographs, obtained with transmission electron microscopy, confirmed the vesicular nature of the membranes and revealed that basolateral membrane vesicles generally were smaller and more irregular in shape than brush border membrane vesicles. The vesicular nature of isolated membrane preparations was confirmed with osmotic activity experiments. Enrichment of brush border and basolateral membrane fractions compared to the initial homogenate and the vesicular configuration of both preparations indicate that the isolated brush border and basolateral membrane preparations were suitable models for evaluating nutrient transport properties of bovine small intestine. The number of transport experiments possible per animal using the membrane vesicle technique is many times more efficient than some other in vitro techniques (i.e., intestinal rings or everted sacs).