PREPARATION AND USE OF BOVINE JEJUNAL BRUSH BORDER MEMBRANE VESICLES TO INVESTIGATE AMINO ACID UPTAKE (NUTRIENT, RUMINANT, ABSORPTION, CATTLE, TRANSPORT)

Purified brush border membranes were obtained from homogenized jejunal epithelial cells of slaughtered cattle by divalent cation aggregation of non-brush border membranes and differential centrifugation. Membrane marker enzyme assays determined effectiveness of the fractionation procedure. Compared to the cellular homogenate, maltase and Na('+)-K('+)-ATPase specific activities in the membrane fraction isolated from the interface of discontinuous (35 and 45% w/w) sucrose gradients increased 14.5 (+OR-) 2.0 and 1.9 (+OR-) 0.7 fold while enzyme recoveries averaged 20.2 (+OR-) 3.6 and 2.4 (+OR-) 1.0%. These data indicate significant enrichment in brush border membranes with minimal basolateral membrane contamination. Vesicles formed from this membrane fraction had a predominately (93 (+OR-) 2%) luminal side out orientation. After incubating vesicles with radiolabelled substrates, vesicles and accumulated substrates were separated from the incubation buffer by filtration and substrate uptake was 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. Vesicles exhibited sodium 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. Results indicate that these vesicles can be utilized to investigate nutrient uptake by the bovine small intestine. Inhibition studies were conducted under conditions shown to provide linear rates of accumulation. Inhibition of alanine uptake increased with increasing extravesicular inhibitor concentration until a plateau value was reached. Inhibition of sodium dependent alanine uptake by 100 mM glycine was 72% while 25 mM isoleucine, valine or methionine completely inhibited initial alanine uptake. These results indicate the existence of at least two sodium dependent transport systems, one which can and one which cannot transport glycine. Equimolar mixtures of 20 L-amino acids inhibited alanine uptake at concentrations more representative of the free amino acids content of intestinal digesta, suggesting that significant interaction among amino acids for uptake may occur under in vivo conditions.