Bazzi M D, Nelsestuen G L
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1991 Aug 13;30(32):7961-9. doi: 10.1021/bi00246a013.
Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)
从牛脑中纯化出的蛋白激酶C以及另外两种分子量分别为64 kDa和32 kDa的蛋白质，构成了一种能以磷脂依赖方式结合大量钙离子的蛋白质类型。本研究表明，这些蛋白质还能诱导膜中酸性磷脂的广泛聚集。酸性磷脂的聚集通过附着在酸性磷脂（磷脂酸或磷脂酰甘油）上的荧光探针的自猝灭来检测。将这些蛋白质添加到含有15%荧光标记磷脂酸并分散在中性磷脂酰胆碱中的磷脂囊泡中，会导致荧光信号广泛、快速且依赖于钙的猝灭。荧光猝灭要求与蛋白质 - 膜结合特性相符。正如预期的那样，将这些蛋白质添加到含有大量过量酸性磷脂分散的荧光磷脂的磷脂囊泡中，只会产生微小的荧光变化。此外，这些蛋白质与由100%荧光磷脂组成的囊泡结合不会导致荧光猝灭。蛋白质与含有5 - 50%荧光磷脂的囊泡结合表现出不同程度的荧光猝灭，这与囊泡外层酸性磷脂广泛分离所预期的行为非常相似。因此，荧光猝灭似乎是由于蛋白质 - 膜结合时聚集的荧光团的自猝灭导致的。这些结果与蛋白质 - 膜结合一致，这种结合是由蛋白质与膜中酸性磷脂之间的钙桥维持的。由于每种蛋白质在磷脂存在下能结合八个或更多钙离子，它们可能各自诱导了相关数量酸性磷脂的聚集。（摘要截短于250字）
