Characterization of an atypical lipoprotein-binding protein in human aortic media membranes by ligand blotting.
By use of ligand-blotting techniques, this study investigated lipoprotein-binding proteins in human aortic smooth muscle. PAGE was performed under non-reducing conditions, and, using low-density lipoprotein (LDL) as ligand, with rabbit anti-apolipoprotein (apo) B and 125I-labelled goat anti-rabbit IgG as primary and secondary antibodies respectively, we demonstrate that membranes from human aortic media (and cultured human smooth-muscle cells) contain a major lipoprotein-binding protein with an apparent molecular mass of 105 kDa. Anionized preparations (carbamoyl- and acetyl-) of LDL, which did not displace 125I-LDL bound to the apo B,E receptor of cultured fibroblasts, were also recognized as ligands for the 105 kDa protein in aortic media membranes. LDL binding to 105 kDa protein was decreased in the presence of high density lipoprotein (HDL), although more than 100-fold molar excess of HDL was required to achieve 50% displacement of bound LDL. The LDL-binding activity of 105 kDa protein was inhibited by EDTA, and was also significantly decreased when samples were reduced by beta-mercaptoethanol before electrophoresis. Monoclonal antibodies against apo B,E receptor reacted with partially purified bovine adrenal apo B,E receptor, but not with 105 kDa protein of human aortic media membranes. The spectrum of properties of this vascular smooth-muscle lipoprotein-binding protein binding are clearly distinct from those of other previously characterized lipoprotein-binding molecules.
Kuzmenko
Y S
YS
Laboratory of Molecular Endocrinology, Cardiology Research Center, Moscow, Russia.
Bochkov
V N
VN
Philippova
M P
MP
Tkachuk
V A
VA
Resink
T J
TJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000906:Antibodies; D001013:Aorta, Thoracic / Q000378:metabolism*; D015153:Blotting, Western; D002122:Calcium Chloride / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006041:Goats / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood / Q000302:isolation & purification; D008875:Middle Aged; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D012867:Skin / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D008075:Lipoproteins, HDL; D004492:Edetic Acid; D002122:Calcium Chloride