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 the ratio of luminal to abluminal membrane in each fraction of the gradient. The results of this analysis indicate that gamma-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 Na+/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.
Sánchez del Pino
M M
MM
Department of Physiology and Biophysics, Finch University of Health Sciences, Chicago Medical School, Illinois 60064, USA.
Hawkins
R A
RA
Peterson
D R
DR
D015720:5'-Nucleotidase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015415:Biomarkers; D001812:Blood-Brain Barrier; D002196:Capillaries / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002501:Centrifugation, Zonal; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D005362:Ficoll; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008833:Microcirculation / Q000378:metabolism* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010042:Ouabain / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D015415:Biomarkers; D007527:Isoenzymes; D005362:Ficoll; D010042:Ouabain; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase