Sapirstein V S, Nolan C, Stern R, Ciocci M, Masur S K
Division of Molecular Biology, Nathan Kline Institute, Orangeburg, NY 10962.
J Neurochem. 1988 Sep;51(3):925-33. doi: 10.1111/j.1471-4159.1988.tb01829.x.
We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.
我们分析了脑包被小泡和突触质膜中质膜蛋白脂蛋白的存在情况。从小牛脑灰质中分离出包被小泡，最后在Sephacryl S - 1000上进行纯化，并通过色谱法重新分离两次以确保均一性。通过凝胶电泳、网格蛋白重链免疫印迹和电子显微镜对各组分进行分析。使用免疫印迹分析，我们能够证明这些包被小泡中存在显著水平的质膜蛋白脂蛋白（即，约占这些小泡双层蛋白的1%）。包被小泡的重新分离并没有降低该组分中蛋白质的浓度。去除网格蛋白包被蛋白或使包被小泡暴露于0.1 M Na2CO3表明，质膜蛋白脂蛋白在脱包被和裂解过程中不会被去除，而是这些小泡膜双层的固有成分。这些研究表明，质膜蛋白脂蛋白占包被小泡双层蛋白的相当一部分，这表明这些小泡可能是质膜蛋白脂蛋白在细胞内运输的载体。从成年大鼠脑中分离突触质膜蛋白脂蛋白，并使用免疫印迹法估计质膜蛋白脂蛋白的含量，证实了早期的研究，即该蛋白在该膜组分中也大量存在（约1 - 2%）。该蛋白在突触质膜中的水平表明，突触质膜是这些小泡可能靶向的主要部位之一，或者是该蛋白被回收的部位。
