Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies.
Studies were conducted to identify and localize albumin binding proteins (ABPs) in endothelial cells using rabbits polyclonal anti-idiotypic antibodies (Ab2) raised against the affinity-purified anti-bovine serum albumin IgG. Ab2 were purified by fast performance liquid chromatography. The anti-idiotypic nature of the IgG was assessed by (i) the capacity to inhibit albumin binding to its specific antibody in a dose-dependent manner, (ii) the lack of interaction with albumin, and (iii) the interaction with anti-albumin antibodies of diverse origins. The latter characteristic indicates that although polyclonal, the purified anti-idiotypic antibodies contain some of the Ab2 beta type. The binding of Ab2 to cultured bovine aortic endothelial cell surfaces was saturable and specific as demonstrated by radioimmunoassay (RIA) and immunofluorescence studies respectively. A competitive RIA was used to test whether Ab2 competed for albumin binding to bovine aortic endothelial cells (BAECs) (presumably to ABPs). It was found that Ab2 inhibited binding of [125I]albumin to BAECs in a dose-dependent fashion. Immunoblot analysis of extracts of BAECs, microvascular endothelial cells, and lung showed that both Ab2 and albumin bind specifically to two polypeptides with an apparent molecular mass of 18 and 31 kDa. In addition, upon radioiodination of BAECs apical membrane proteins, Ab2 bound specifically and immunoprecipitated restrictively two radiolabeled cell surface proteins of 18 and 31 kDa. The results provide direct evidence for the presence of the 18 and 31 kDa peptides (ABPs) on the endothelial cell membrane and/or associated structures, i.e. open plasmalemmal vesicles and uncoated pits.
Dobrila
L
L
Institute of Cellular Biology and Pathology, Bucharest, Romania.
Antohe
F
F
Heltianu
C
C
Simionescu
M
M
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D004730:Endothelium, Vascular / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D012709:Serum Albumin / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D002352:Carrier Proteins; D008565:Membrane Proteins; D012709:Serum Albumin