Kuzmenko Y S, Bochkov V N, Philippova M P, Tkachuk V A, Resink T J
Laboratory of Molecular Endocrinology, Cardiology Research Center, Moscow, Russia.
Biochem J. 1994 Oct 1;303 ( Pt 1)(Pt 1):281-7. doi: 10.1042/bj3030281.
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.
本研究利用配体印迹技术，对人主动脉平滑肌中的脂蛋白结合蛋白进行了研究。在非还原条件下进行聚丙烯酰胺凝胶电泳（PAGE），以低密度脂蛋白（LDL）作为配体，分别用兔抗载脂蛋白（apo）B和125I标记的山羊抗兔IgG作为一抗和二抗，我们证明人主动脉中层膜（以及培养的人平滑肌细胞）含有一种主要的脂蛋白结合蛋白，其表观分子量为105 kDa。LDL的阴离子化制剂（氨基甲酰化和乙酰化）不能取代与培养的成纤维细胞的apo B、E受体结合的125I-LDL，但也被认为是主动脉中层膜中105 kDa蛋白的配体。在高密度脂蛋白（HDL）存在的情况下，LDL与105 kDa蛋白的结合减少，尽管需要超过100倍摩尔过量的HDL才能实现50%的结合LDL置换。105 kDa蛋白的LDL结合活性受到乙二胺四乙酸（EDTA）的抑制，并且当样品在电泳前用β-巯基乙醇还原时，其结合活性也显著降低。抗apo B、E受体的单克隆抗体与部分纯化的牛肾上腺apo B、E受体发生反应，但不与人主动脉中层膜的105 kDa蛋白发生反应。这种血管平滑肌脂蛋白结合蛋白结合的特性谱与其他先前表征的脂蛋白结合分子的特性谱明显不同。
