Characterization of the Blood‐Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Abstract

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.

Transport of Human Immunodeficiency Virus Type 1 Pseudoviruses across the Blood-Brain Barrier: Role of Envelope Proteins and Adsorptive Endocytosis

“…The radioactivity in these tubes was taken to represent binding to the surface of the IBM that was reversible. Acid-washed microvessels were lysed according to the method of Lidinsky and Drewes (41). Acid-washed microvessel pellets were resuspended in 400 l of distilled water containing 1% BSA and agitated for 2 h at 4°C.…”

Human blood-brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels.

“…The final capillary pellet was suspended in a cryopreservation buffer (0.02 M Tris, 0.25 M sucrose, and 2 mM dithiothreitol) and stored at Ϫ 70 Њ C under liquid nitrogen. Human brain capillary membranes were isolated from the capillaries of the female donor on the same day as the capillary isolation according to the method of Lidinsky and Drewes (29). Both capillaries and plasma membranes isolated by these techniques maintain biochemical characteristics of the intact BBB, as characterized previously in this laboratory for the human BBB insulin receptor (21).…”

Identification and characterization of the glucose transporter of the blood-brain barrier by cytochalasin B binding and immunological reactivity.

“…In two experiments, subfractions of pig cerebral microvessels were obtained by the method of Lidinsky and Drewes (24) with modifications. Microvessels were suspended in 0.01 M Tris buffer (pH 7.4) and stirred continuously for 2 hr.…”