Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.
Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)
Antohe
F
F
Institute of Cellular Biology and Pathology, Bucharest/Romania.
Heltianu
C
C
Simionescu
M
M
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D004822:Epididymis / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D005455:Fluorescent Antibody Technique; D008297:Male; D008833:Microcirculation / Q000737:chemistry / Q000276:immunology; D016253:Microscopy, Immunoelectron; D009206:Myocardium / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D051381:Rats; D017457:Receptors, Albumin; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000302:isolation & purification
D017457:Receptors, Albumin; D011956:Receptors, Cell Surface