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 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.
Highlights
From the Department of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064
Na+fK+·ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin
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
Summary
Vol 270, No 25, Issue of June 23, pp. 14907-14912, 1995 Printed in U.S.A. Biochemical Discrimination between Luminal and Abluminal Enzyme and Transport Activities of the Blood-Brain Barrier*. A multiplicity ofisoforms (3 IX and 2 (3 subunits of the Na+/K+-ATPase) has been shown recently using Western blot analysis [13], indicating that six different isoenzymes may be expressed in brain endothelial cells These results suggest that while abluminal membranes contain most of the Na +/K+-ATPase activity, or the most active isofonn, some activity may be present in luminal membranes. We have previously shown that membrane vesicles isolated from bovine brain endothelial cells can be used to study transport [14] These results were interpreted according to the consensus opinion [3] that GGT and alkaline phosphatase were located on the luminal membrane and Na+/K+-ATPase was on the abluminal membrane.
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