Okumura Y, Ishitobi M, Sobel M, Akiyoshi K, Sunamoto J
Division of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Biochim Biophys Acta. 1994 Sep 14;1194(2):335-40. doi: 10.1016/0005-2736(94)90316-6.
The direct transfer of membrane proteins from human platelets to the liposomal fraction was examined, particularly in relation to platelet activation during the process. The incorporation of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), in the interacting liposome considerably enhanced the efficiency of the protein transfer. The transfer proceeded with neither significant activation nor lysis of the platelet, and the activation of the platelet with thrombin did not affect the amount of the transferred proteins. A wide range of platelet membrane proteins was transferred, and they were almost comparable to those in a sample prepared by glycerol lysis/centrifugation. In addition, they included the major surface glycoproteins GPIIb and GPIIIa without noticeable contamination of soluble cytosol proteins. The protein transfer method is a one-pot process and clearly more convenient than the conventional 'extract and reconstitute' approach. These results strongly support the use of the transfer process, especially with DDPC, as an alternative to the conventional detergent-solubilization or the solvent-extraction methods for preparation of samples of platelet membrane proteins.
研究了膜蛋白从人血小板直接转移至脂质体部分的情况，尤其涉及该过程中血小板的激活。在相互作用的脂质体中加入人工边界脂质1,2 - 二肉豆蔻酰氨基 - 1,2 - 脱氧磷脂酰胆碱（DDPC），可显著提高蛋白质转移效率。转移过程中血小板既无明显激活也无裂解，用凝血酶激活血小板并不影响转移蛋白的量。多种血小板膜蛋白被转移，且与通过甘油裂解/离心制备的样品中的蛋白几乎相当。此外，它们包括主要表面糖蛋白GPIIb和GPIIIa，几乎没有可溶性胞质溶胶蛋白的明显污染。蛋白质转移方法是一个一步法过程，明显比传统的“提取并重构”方法更方便。这些结果有力地支持了使用转移过程，特别是使用DDPC，作为传统去污剂增溶或溶剂萃取方法的替代方法来制备血小板膜蛋白样品。
