Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.
Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.
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胶体金-低密度脂蛋白缀合物作为膜受体探针。
DOI：
10.1073/pnas.78.1.368
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发表时间：
1981
影响因子：
11.1
通讯作者：
Chien,S
中科院分区：
文献类型：
--
作者：
Handley,DA;Arbeeny,CM;Witte,LD;Chien,S
我们开发了一种将低密度脂蛋白 (LDL) 与胶体金结合的方法。 1 分钟后，LDL 静电吸附到带负电的金颗粒上，从而完成结合。每个结合物由大约八个生物活性 LDL 分子组成，聚集在中央 19 nm 金颗粒周围。酸性 (pH 4)、碱性 (pH 9) 或高离子 (600 毫渗氯化钠) 环境不会使缀合物解离。胶体金是一种电子密集、不可降解的标记物，很容易在细胞内识别，可作为研究受体结合和内吞作用的有价值的探针。通过使用改进的钌红染色方法，当结合物的 LDL 分子与细胞表面受体结合时，可以直接观察到它们。培养的人成纤维细胞对缀合物的受体结合（4 摄氏度）表明金颗粒位于距离膜的包被凹坑区域 18-21 nm 处。该距离与 LDL 的直径相似，表明在囊泡内吞作用和早期溶酶体掺入（37°C 10 分钟）过程中受体同时内化。持续的内化（37°C 下 30-60 分钟）导致溶酶体内形成游离金池。
We have developed a method for conjugating low density lipoproteins (LDL) with colloidal gold. Conjugation, complete after 1 min, occurs by electrostatic adsorption of the LDL to the negatively charged gold particle. Each conjugate consists of approximately eight biologically active LDL molecules clustered around a central 19-nm gold granule. Acidic (pH 4), alkaline (pH 9), or high ionic (600 milliosmolar NaCl) environments do not dissociate the conjugate. Colloidal gold is an electron-dense, nondegradable marker that is easily identified within the cell and serves as a valuable probe for studying receptor binding and endocytosis. By using a modified method of ruthenium red staining, the LDL molecules of the conjugate can be directly visualized when they are bound to the cell surface receptor. Receptor binding (4 degrees C) of the conjugate by cultured human fibroblasts reveals that the gold granule is positioned 18-21 nm from the coated pit region of the membrane. This distance, similar to the diameter of LDL, suggests concomitant internalization of the receptor during vesicular endocytosis and early lysosomal incorporation (10 min at 37 degrees C). Continued internalization (30-60 min at 37 degrees C) results in the formation of free pools of gold within the lysosome.