Identification of albumin-binding proteins in capillary endothelial cells.
Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.
Ghinea
N
N
Institute of Cellular Biology and Pathology, Bucharest, Romania.
Fixman
A
A
Alexandru
D
D
Popov
D
D
Hasu
M
M
Ghitescu
L
L
Eskenasy
M
M
Simionescu
M
M
Simionescu
N
N
D000273:Adipose Tissue / Q000098:blood supply; D000418:Albumins / Q000378:metabolism*; D000818:Animals; D001011:Aorta; D002196:Capillaries; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000032:analysis*; D007118:Immunoassay; D007525:Isoelectric Focusing; D008168:Lung / Q000098:blood supply; D008297:Male; D051379:Mice; D008833:Microcirculation; D011817:Rabbits; D051381:Rats; D017457:Receptors, Albumin; D011956:Receptors, Cell Surface / Q000032:analysis*
D000418:Albumins; D017457:Receptors, Albumin; D011956:Receptors, Cell Surface