{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/101336"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/101336","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Preparation of brush border and basolateral membrane vesicles from bovine intestine for nutrient uptake studies","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Wilson, Jonathan Wesley"],"institution":"Virginia Polytechnic Institute and State University","degree_name":"M.S.","degree_level":"masters","degree_discipline":"Animal Science","degree_department":"Animal Science","school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":1986,"date_issued":"1986","date_published":"1986","updated_at":"2026-07-22T22:19:31Z","subjects":[],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10919/101336","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.department","label":"Department","values":["Animal Science"]},{"key":"dc:creator","label":"Author","values":["Wilson, Jonathan Wesley"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2020-12-14T17:48:27Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2020-12-14T17:48:27Z"]},{"key":"dc:date.issued","label":"Date","values":["1986"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Polytechnic Institute and State University"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Animal Science"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M.S."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/101336"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["M.S."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Preparation of brush border and basolateral membrane vesicles from bovine intestine for nutrient uptake studies"]}]}],"canonical_facts":{"dc:contributor.department":["Animal Science"],"dc:creator":["Wilson, Jonathan Wesley"],"dc:date.accessioned":["2020-12-14T17:48:27Z"],"dc:date.available":["2020-12-14T17:48:27Z"],"dc:date.issued":["1986"],"dc:description.abstract":["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. 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