Preparation of brush border membrane vesicles from fresh and frozen XXXX intestine for nutrient uptake studies.

@entity859 @entity1372 @entity5222 @entity972 @entity291
Purified brush border membranes were obtained from homogenized jejunal epithelial cells of @entity291 by divalent cation aggregation of nonbrush border membranes and differential centrifugation. Membrane marker enzyme assays determined effectiveness of the fractionation procedure. Compared with cellular homogenate, maltase and @entity1372 +- @entity972 +- @entity859 triphosphatase specific activities in the membrane fraction isolated from the interface of discontinuous (35 and 45% wt/wt) sucrose gradients increased 14.5- and 1.9-fold whereas enzyme recoveries averaged 20.2 and 2.4%. These data indicate significant enrichment in brush border membranes with minimal basolateral membrane contamination. @entity5222 formed from this membrane fraction had a predominately (93%) luminal side out orientation. After incubating vesicles with radiolabeled substrates, vesicles and accumulated substrates were separated from the incubation buffer by filtration and substrate uptake quantified by liquid scintillation counting. Observed uptake was the result of substrate accumulation within an osmotically active intravesicular space and was not due to nonspecific binding of the substrate to vesicular membranes. @entity5222 exhibited @entity1372 -dependent and independent substrate uptake pathways and were able to discriminate between substrate stereoisomers for uptake. Major differences were not detected between results obtained with vesicles prepared from fresh or frozen intestines. These vesicles can be utilized to investigate nutrient uptake by the @entity291 small intestine.
