Browsing by Author "Wilson, Jonathan Wesley"

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    Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles
    Wilson, Jonathan Wesley (Virginia Polytechnic Institute and State University, 1988)
    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. Membrane marker enzymes were used to determine the effectiveness of the fractionation procedure. Alkaline phosphatase and sodium-potassium adenosine triphosphatase served as the marker enzymes for the brush border and basolateral membranes, respectively. The brush border fraction was enriched 5.1-fold over the cellular homogenate. Purification of 10.1-fold over cellular homogenate was obtained for the basolateral membrane fraction. Electronmicrographs and osmotic response data were used to confirm the vesicular nature of the membrane preparations. Brush border membrane vesicles from bovine jejunal and ileal tissue were used to evaluate lysine (LYS) and methionine (MET) transport. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium-dependent (Na⁺) and sodium independent (Na⁻) systems. Total LYS and MET uptake by ileal tissue tended to be higher than jejunal tissue at all concentrations evaluated but differences were significant (P<.O5) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal tissue appeared to be due to the Na⁺ component of LYS uptake and the diffusion component of MET uptake. Methionine transporters had lower affinities and higher capacities than the corresponding LYS transporters in both ileal and jejunal tissue. Methionine transport was greater (P<.O5) than LYS transport in both ileal and jejunal tissue when initial amino acid concentration was 7.5 mM. When initial amino acid concentration was 1.25 mM, MET uptake was greater (P<.13) than LYS uptake in jejunal but not ileal tissue. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent of substrate concentration.
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    Preparation of brush border and basolateral membrane vesicles from bovine intestine for nutrient uptake studies
    Wilson, Jonathan Wesley (Virginia Polytechnic Institute and State University, 1986)
    Brush border and basolateral membrane vesicles were isolated by subjecting homogenized mucosal cells from bovine small intestine to a divalent cation aggregation followed by a series of differential and density gradient centrifugations. Membrane marker enzyme assays were used to monitor the effectiveness of the fractionation procedure. Enrichments were determined by comparing the enzyme specific activities of the membrane fractions to the homogenate. Sodium-potassium adenosine triphosphatase and alkaline phosphatase served as the enzyme markers for the basolateral and brush border membranes, respectively. Basolateral membrane vesicles enriched 11.1 fold were isolated from the interface of the 31 and 34% sucrose bands of a discontinuous sucrose gradient. Brush border membranes enriched 10.1 fold were isolated from the surface of the 28% sucrose band of a discontinuous sucrose gradient. The use of frozen rather than fresh mucosal tissue in the isolation procedures was found to enhance the purification of basolateral and brush border membrane fractions. The transport capabilities of vesicles were demonstrated by incubating vesicles with radiolabeled substrate, then separating the vesicles and transported substrate from the incubation buffer by filtration. Substrate uptakes were quantified by liquid scintillation counting. Basolateral membrane vesicles were observed to accumulate substrate into an osmotically active space and to have Na⁺-dependent alanine transport capabilities. The use of basolateral and brush border membrane vesicles as tools to investigate nutrient uptake allows the investigator to manipulate both the extravesicular and intravesicular environments, thus making possible the evaluation of the complex interactions which are involved in nutrient transport mechanisms.