Newton D E, Shaw J M
Biochim Biophys Acta. 1984 Jul 6;794(2):194-205. doi: 10.1016/0005-2760(84)90146-2.
Binding studies were performed with bovine adrenal cortex membranes, human 125I-labelled high-density lipoprotein (HDL) and modified photoactivable derivatives of 125I-labelled HDL, namely 125I-labelled HDL-amidinophenylazide and 125I-labelled HDL-amidopropionyldithiophenylazide. The purity of the apolipoprotein composition of the 125I-labelled HDL and photoactivable 125I-labelled HDL used in the binding studies was determined by Coomassie blue and silver staining, and by measuring 125I-labelled cpm after SDS-polyacrylamide gel electrophoresis. About 45% of the 125I-labelled HDL binding to the membranes occurred in the presence of excess EDTA and only unlabelled HDL competed for the binding site. The 125I-labelled interaction with this binding site on the membranes did not require calcium. In addition, 40% of the 125I-labelled HDL binding was to an EDTA-sensitive site, and unlabelled HDL and low-density lipoprotein (LDL) competed for the binding site. Consequently, adrenal cortex membranes have binding sites which show cross reactivity for both HDL and LDL. Modification of 58% of the apolipoprotein lysine residues of 125I-labelled HDL with methylazidophenylimidate, a reagent which maintains the positive charge at lysine residues, had little affect on binding to EDTA-sensitive and insensitive sites. In contrast, modification of 35% of apolipoprotein lysine residues of 125I-labelled HDL with N-succinimidyl(4-azidophenyldithio)propionate, a reagent which converts charged amino lysines to amide bonds, showed binding properties which were almost totally inhibited by EDTA.
采用牛肾上腺皮质膜、人125I标记的高密度脂蛋白（HDL）以及125I标记HDL的修饰光活化衍生物，即125I标记的HDL-脒基苯基叠氮化物和125I标记的HDL-酰胺丙酰二硫代苯基叠氮化物进行结合研究。结合研究中使用的125I标记HDL和光活化125I标记HDL的载脂蛋白成分纯度通过考马斯亮蓝染色、银染色以及SDS-聚丙烯酰胺凝胶电泳后测量125I标记的每分钟计数来确定。在过量乙二胺四乙酸（EDTA）存在的情况下，约45%的125I标记HDL与膜结合，且只有未标记的HDL竞争结合位点。125I标记物与膜上该结合位点的相互作用不需要钙。此外，40%的125I标记HDL结合到一个对EDTA敏感的位点，未标记的HDL和低密度脂蛋白（LDL）竞争该结合位点。因此，肾上腺皮质膜具有对HDL和LDL均表现出交叉反应性的结合位点。用甲基叠氮基苯基亚氨酸酯（一种在赖氨酸残基处保持正电荷的试剂）修饰125I标记HDL的58%载脂蛋白赖氨酸残基，对其与EDTA敏感和不敏感位点的结合影响不大。相比之下，用N-琥珀酰亚胺基（4-叠氮苯基二硫代）丙酸酯（一种将带电荷的氨基赖氨酸转化为酰胺键的试剂）修饰125I标记HDL的35%载脂蛋白赖氨酸残基，其结合特性几乎完全被EDTA抑制。
