Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles
The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show tha...
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Veröffentlicht in: | Biochemical journal 1990-08, Vol.269 (3), p.565-571 |
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description | The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides. |
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Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.</description><identifier>ISSN: 0264-6021</identifier><identifier>EISSN: 1470-8728</identifier><identifier>DOI: 10.1042/bj2690565</identifier><identifier>PMID: 2167659</identifier><language>eng</language><publisher>England</publisher><subject>Amino Acids - pharmacology ; Animals ; Biological Transport ; Deoxyribonucleases - metabolism ; Dipeptides - pharmacokinetics ; Dipeptides - pharmacology ; enterocytes ; Enzyme Activation ; Glucose - metabolism ; Intestinal Mucosa - metabolism ; Intestines - cytology ; Intestines - ultrastructure ; Intracellular Membranes - metabolism ; Kinetics ; Membrane Proteins - immunology ; membrane vesicles ; Microvilli - immunology ; Microvilli - ultrastructure ; Osmolar Concentration ; Ouabain - pharmacology ; Phosphoric Monoester Hydrolases - metabolism ; Potassium - pharmacology ; Rabbits</subject><ispartof>Biochemical journal, 1990-08, Vol.269 (3), p.565-571</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c467t-c0c10fd604082b1935ec1a373e58812e0d230bccbbbaed2705b104d30147fcd63</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1131624/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1131624/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2167659$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dyer, J</creatorcontrib><creatorcontrib>Beechey, R B</creatorcontrib><creatorcontrib>Gorvel, J P</creatorcontrib><creatorcontrib>Smith, R T</creatorcontrib><creatorcontrib>Wootton, R</creatorcontrib><creatorcontrib>Shirazi-Beechey, S P</creatorcontrib><title>Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles</title><title>Biochemical journal</title><addtitle>Biochem J</addtitle><description>The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.</description><subject>Amino Acids - pharmacology</subject><subject>Animals</subject><subject>Biological Transport</subject><subject>Deoxyribonucleases - metabolism</subject><subject>Dipeptides - pharmacokinetics</subject><subject>Dipeptides - pharmacology</subject><subject>enterocytes</subject><subject>Enzyme Activation</subject><subject>Glucose - metabolism</subject><subject>Intestinal Mucosa - metabolism</subject><subject>Intestines - cytology</subject><subject>Intestines - ultrastructure</subject><subject>Intracellular Membranes - metabolism</subject><subject>Kinetics</subject><subject>Membrane Proteins - immunology</subject><subject>membrane vesicles</subject><subject>Microvilli - immunology</subject><subject>Microvilli - ultrastructure</subject><subject>Osmolar Concentration</subject><subject>Ouabain - pharmacology</subject><subject>Phosphoric Monoester Hydrolases - metabolism</subject><subject>Potassium - pharmacology</subject><subject>Rabbits</subject><issn>0264-6021</issn><issn>1470-8728</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFKxDAQhoMo67p68AGEngQP1UnaJu1FkEVXYcWLnkOSTrVL2qxJd6Fvb8Rl0ZOnYZiPn2_4CTmncE0hZzd6xXgFBS8OyJTmAtJSsPKQTIHxPOXA6DE5CWEFQHPIYUImjHLBi2pKnhd2NKNNl-naO9v2mAxe9WHt_JC0feKV1u2QYD-gd2YcMNEqOKviqmzaYacjjckWQ2sshlNy1Cgb8Gw3Z-Tt4f51_pguXxZP87tlanIuhtSAodDUPLqUTNMqK9BQlYkMi7KkDKFmGWhjtNYKayag0PHNOov6ojE1z2bk9id3vdEd1ib6RR-59m2n_CidauXfS99-yHe3lZRmlLM8BlzuArz73GAYZNcGg9bGb9wmSFFVPLr9D9JCMEaBRfDqBzTeheCx2dtQkN8lyX1Jkb34rb8nd61kX8pRjj0</recordid><startdate>19900801</startdate><enddate>19900801</enddate><creator>Dyer, J</creator><creator>Beechey, R B</creator><creator>Gorvel, J P</creator><creator>Smith, R T</creator><creator>Wootton, R</creator><creator>Shirazi-Beechey, S P</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19900801</creationdate><title>Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles</title><author>Dyer, J ; Beechey, R B ; Gorvel, J P ; Smith, R T ; Wootton, R ; Shirazi-Beechey, S P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c467t-c0c10fd604082b1935ec1a373e58812e0d230bccbbbaed2705b104d30147fcd63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Amino Acids - pharmacology</topic><topic>Animals</topic><topic>Biological Transport</topic><topic>Deoxyribonucleases - metabolism</topic><topic>Dipeptides - pharmacokinetics</topic><topic>Dipeptides - pharmacology</topic><topic>enterocytes</topic><topic>Enzyme Activation</topic><topic>Glucose - metabolism</topic><topic>Intestinal Mucosa - metabolism</topic><topic>Intestines - cytology</topic><topic>Intestines - ultrastructure</topic><topic>Intracellular Membranes - metabolism</topic><topic>Kinetics</topic><topic>Membrane Proteins - immunology</topic><topic>membrane vesicles</topic><topic>Microvilli - immunology</topic><topic>Microvilli - ultrastructure</topic><topic>Osmolar Concentration</topic><topic>Ouabain - pharmacology</topic><topic>Phosphoric Monoester Hydrolases - metabolism</topic><topic>Potassium - pharmacology</topic><topic>Rabbits</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dyer, J</creatorcontrib><creatorcontrib>Beechey, R B</creatorcontrib><creatorcontrib>Gorvel, J P</creatorcontrib><creatorcontrib>Smith, R T</creatorcontrib><creatorcontrib>Wootton, R</creatorcontrib><creatorcontrib>Shirazi-Beechey, S P</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dyer, J</au><au>Beechey, R B</au><au>Gorvel, J P</au><au>Smith, R T</au><au>Wootton, R</au><au>Shirazi-Beechey, S P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles</atitle><jtitle>Biochemical journal</jtitle><addtitle>Biochem J</addtitle><date>1990-08-01</date><risdate>1990</risdate><volume>269</volume><issue>3</issue><spage>565</spage><epage>571</epage><pages>565-571</pages><issn>0264-6021</issn><eissn>1470-8728</eissn><abstract>The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.</abstract><cop>England</cop><pmid>2167659</pmid><doi>10.1042/bj2690565</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Amino Acids - pharmacology Animals Biological Transport Deoxyribonucleases - metabolism Dipeptides - pharmacokinetics Dipeptides - pharmacology enterocytes Enzyme Activation Glucose - metabolism Intestinal Mucosa - metabolism Intestines - cytology Intestines - ultrastructure Intracellular Membranes - metabolism Kinetics Membrane Proteins - immunology membrane vesicles Microvilli - immunology Microvilli - ultrastructure Osmolar Concentration Ouabain - pharmacology Phosphoric Monoester Hydrolases - metabolism Potassium - pharmacology Rabbits |
title | Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles |
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