Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (∗)
Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as...
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| Veröffentlicht in: | The Journal of biological chemistry 1995-06, Vol.270 (25), p.14907-14912 |
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| container_title | The Journal of biological chemistry |
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| creator | del Pino, Manuel M. Sánchez Hawkins, Richard A. Peterson, Darryl R. |
| description | Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that g-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5′-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Nag/K+-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K+-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations. |
| doi_str_mv | 10.1074/jbc.270.25.14907 |
| format | Article |
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Sánchez ; Hawkins, Richard A. ; Peterson, Darryl R.</creator><creatorcontrib>del Pino, Manuel M. Sánchez ; Hawkins, Richard A. ; Peterson, Darryl R.</creatorcontrib><description>Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that g-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5′-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Nag/K+-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K+-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.270.25.14907</identifier><identifier>PMID: 7797469</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>5'-Nucleotidase - metabolism ; Alkaline Phosphatase - metabolism ; Animals ; Biological Transport ; Biomarkers ; Blood-Brain Barrier ; Capillaries - metabolism ; Capillaries - ultrastructure ; Cattle ; Cell Fractionation ; Cell Membrane - metabolism ; Cell Membrane - ultrastructure ; Centrifugation, Zonal ; Endothelium, Vascular - metabolism ; Endothelium, Vascular - ultrastructure ; Ficoll ; gamma-Glutamyltransferase - metabolism ; Isoenzymes - metabolism ; Kinetics ; Microcirculation - metabolism ; Microcirculation - ultrastructure ; Microscopy, Electron, Scanning ; Ouabain - pharmacology ; Sodium-Potassium-Exchanging ATPase - metabolism</subject><ispartof>The Journal of biological chemistry, 1995-06, Vol.270 (25), p.14907-14912</ispartof><rights>1995 © 1995 ASBMB. 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Sánchez</creatorcontrib><creatorcontrib>Hawkins, Richard A.</creatorcontrib><creatorcontrib>Peterson, Darryl R.</creatorcontrib><title>Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (∗)</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that g-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5′-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Nag/K+-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K+-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.</description><subject>5'-Nucleotidase - metabolism</subject><subject>Alkaline Phosphatase - metabolism</subject><subject>Animals</subject><subject>Biological Transport</subject><subject>Biomarkers</subject><subject>Blood-Brain Barrier</subject><subject>Capillaries - metabolism</subject><subject>Capillaries - ultrastructure</subject><subject>Cattle</subject><subject>Cell Fractionation</subject><subject>Cell Membrane - metabolism</subject><subject>Cell Membrane - ultrastructure</subject><subject>Centrifugation, Zonal</subject><subject>Endothelium, Vascular - metabolism</subject><subject>Endothelium, Vascular - ultrastructure</subject><subject>Ficoll</subject><subject>gamma-Glutamyltransferase - metabolism</subject><subject>Isoenzymes - metabolism</subject><subject>Kinetics</subject><subject>Microcirculation - metabolism</subject><subject>Microcirculation - ultrastructure</subject><subject>Microscopy, Electron, Scanning</subject><subject>Ouabain - pharmacology</subject><subject>Sodium-Potassium-Exchanging ATPase - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kE1uFDEQhS0ECkNgzwbJC4Rg0YPd3bbb2c0k4UcaiU2Q2Fn-qWYcdbcH25NoOEFuwP04CU56xAKJ2lhV9d5T-UPoJSVLSkT7_trYZS3IsmZL2koiHqEFJV1TNYx-e4wWhNS0kjXrnqJnKV2TUq2kJ-hECClaLhfosPbBbmH0Vg_4wicb_egnnX2YsIF8CzDhzf5-NGA9Obwyw7G7nH4eRngYXkU9pV2IGa9s9jc-e0g49DhvAa-HEFy1jtpPeK1j9BDx2993v949R096PSR4cXxP0dcPl1fnn6rNl4-fz1ebyrZdnStJbUupkYZ3UveUW1m3jewZ401tpdOs4Yw7qzloqTvW9bTRrhNt74hxlJvmFL2Zc3cx_NhDymos34Rh0BOEfVJCNJzzlhchmYU2hpQi9GpXYOh4UJSoe9qq0FaFtqqZeqBdLK-O2XszgvtrOOIt-9fzfuu_b299BGVm3P_GnM0yKBxuCiGVrIfJgisWm5UL_v83_AHGxJxO</recordid><startdate>19950623</startdate><enddate>19950623</enddate><creator>del Pino, Manuel M. Sánchez</creator><creator>Hawkins, Richard A.</creator><creator>Peterson, Darryl R.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><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>7X8</scope></search><sort><creationdate>19950623</creationdate><title>Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (∗)</title><author>del Pino, Manuel M. Sánchez ; Hawkins, Richard A. ; Peterson, Darryl R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c482t-91c411b9b689af16c92439f55632c9da53656dca6ea9a858f13ad874fd0bd16b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>5'-Nucleotidase - metabolism</topic><topic>Alkaline Phosphatase - metabolism</topic><topic>Animals</topic><topic>Biological Transport</topic><topic>Biomarkers</topic><topic>Blood-Brain Barrier</topic><topic>Capillaries - metabolism</topic><topic>Capillaries - ultrastructure</topic><topic>Cattle</topic><topic>Cell Fractionation</topic><topic>Cell Membrane - metabolism</topic><topic>Cell Membrane - ultrastructure</topic><topic>Centrifugation, Zonal</topic><topic>Endothelium, Vascular - metabolism</topic><topic>Endothelium, Vascular - ultrastructure</topic><topic>Ficoll</topic><topic>gamma-Glutamyltransferase - metabolism</topic><topic>Isoenzymes - metabolism</topic><topic>Kinetics</topic><topic>Microcirculation - metabolism</topic><topic>Microcirculation - ultrastructure</topic><topic>Microscopy, Electron, Scanning</topic><topic>Ouabain - pharmacology</topic><topic>Sodium-Potassium-Exchanging ATPase - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>del Pino, Manuel M. Sánchez</creatorcontrib><creatorcontrib>Hawkins, Richard A.</creatorcontrib><creatorcontrib>Peterson, Darryl R.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>del Pino, Manuel M. Sánchez</au><au>Hawkins, Richard A.</au><au>Peterson, Darryl R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (∗)</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1995-06-23</date><risdate>1995</risdate><volume>270</volume><issue>25</issue><spage>14907</spage><epage>14912</epage><pages>14907-14912</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Luminal and abluminal membrane vesicles derived from bovine brain endothelial cells, the site of the blood-brain barrier, were fractionated in a discontinuous Ficoll gradient. A mathematical analysis was developed to determine the membrane distribution of membrane marker enzyme activities as well as the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that g-glutamyl transpeptidase and amino acid transport system A are located on the luminal and abluminal membranes, respectively. Conversely, 5′-nucleotidase and alkaline phosphatase activities are evenly distributed between both membranes. Although Nag/K+-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin. Na+/K+-ATPase activities associated with each membrane showed different ouabain sensitivities, suggesting that different isoenzymes are located in luminal and abluminal membranes. The analytical procedure used in this study provides a quantitative means to determine the distribution of marker enzymes and transport proteins in partially purified membrane vesicle populations.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>7797469</pmid><doi>10.1074/jbc.270.25.14907</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1995-06, Vol.270 (25), p.14907-14912 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | 5'-Nucleotidase - metabolism Alkaline Phosphatase - metabolism Animals Biological Transport Biomarkers Blood-Brain Barrier Capillaries - metabolism Capillaries - ultrastructure Cattle Cell Fractionation Cell Membrane - metabolism Cell Membrane - ultrastructure Centrifugation, Zonal Endothelium, Vascular - metabolism Endothelium, Vascular - ultrastructure Ficoll gamma-Glutamyltransferase - metabolism Isoenzymes - metabolism Kinetics Microcirculation - metabolism Microcirculation - ultrastructure Microscopy, Electron, Scanning Ouabain - pharmacology Sodium-Potassium-Exchanging ATPase - metabolism |
| title | Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier (∗) |
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