Rakowska M, Zborowski J, Corazzi L
Institute of Biochemistry and Medical Chemistry, University of Perugia, Italy.
J Membr Biol. 1994 Oct;142(1):35-42. doi: 10.1007/BF00233381.
The procedures for purification and reconstitution of rat brain microsomal membrane protein that causes fusion of liposomes at acidic pH are described. A 1,860-fold purification was achieved, starting from the detergent-solubilized microsomal membranes. The fusion process was assayed spectrofluorimetrically by monitoring the formation of terbium-dipicolinic acid complex (Wilschut, J. et al. 1980. Biochemistry 19:6011-6021) evoked by the protein after mixing of two populations of liposomes. The fusogenic activity of the protein inserted into the membrane of Tb3+-containing vesicles was found to be strongly dependent on phospholipid composition and was higher in vesicles enriched with exogenous phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine than in those prepared with an excess of phosphatidylcholine. The vesicles enriched in negatively charged phospholipids were bound to Concanavalin A coupled to Sepharose-4B and could be released from this column only in the presence of a high concentration of alpha-methylmannopyranoside and detergent, indicating a glycoprotein nature of the fusogenic protein. Furthermore, these data show that protein inserted into membrane has its oligosaccharide chains exposed to the environment.
描述了在酸性pH下导致脂质体融合的大鼠脑微粒体膜蛋白的纯化和重组程序。从去污剂溶解的微粒体膜开始，实现了1860倍的纯化。通过监测两群脂质体混合后蛋白质引发的铽 - 二吡啶甲酸络合物的形成（Wilschut，J.等人，1980年。《生物化学》19:6011 - 6021），用荧光分光光度法测定融合过程。发现插入含Tb3 +囊泡膜中的蛋白质的融合活性强烈依赖于磷脂组成，并且在富含外源性磷脂酰丝氨酸、磷脂酰甘油和磷脂酰乙醇胺的囊泡中比在含有过量磷脂酰胆碱制备的囊泡中更高。富含带负电荷磷脂的囊泡与偶联到琼脂糖 - 4B上的伴刀豆球蛋白A结合，并且只有在高浓度的α - 甲基甘露糖苷和去污剂存在下才能从该柱上释放，表明融合蛋白具有糖蛋白性质。此外，这些数据表明插入膜中的蛋白质的寡糖链暴露于环境中。
