ASSOCIATION OF PROTEIN-KINASE-C WITH PHOSPHOLIPID-VESICLES
ASSOCIATION OF PROTEIN-KINASE-C WITH PHOSPHOLIPID-VESICLES
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DOI：
10.1021/bi00375a017
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发表时间：
1987-01-13
期刊：
影响因子：
2.9
通讯作者：
NELSESTUEN, GL
中科院分区：
文献类型：
--
作者：
BAZZI, MD;NELSESTUEN, GL
Ca2+和磷脂依赖性蛋白激酶PKC （protein kinase C， PKC）通过改进的程序从牛脑中纯化，为分析蛋白质-膜结合的物理特性提供了足够数量的稳定蛋白。通过光散射和荧光能量转移测量，研究了PKC与不同组成的磷脂囊泡的结合。低磷脂酰丝氨酸（PS）含量的膜结合特性与外周膜结合一致；PKC显示Ca2+依赖性结合磷脂囊泡含有磷脂酰丝氨酸，磷脂酰肌醇，或磷脂酰甘油。含有0-20% PS（其余磷脂为磷脂酰胆碱）的膜比含有大于20% PS的膜结合的蛋白质少；限制蛋白质与低PS膜结合的因素似乎是酸性磷脂的可用性。PS含量增加20%以上，饱和状态下膜结合蛋白的数量并没有增加，限制因素可能是膜表面蛋白质的立体堆积。膜在空间饱和状态下结合约1g蛋白质/g磷脂。结合具有较高的亲和力（Kd < 5 nM），在实验时间尺度上结合速率较快。乙二醇的加入。-氨基乙醚)-N，N，N‘，N’ -四乙酸与磷脂结合的PKC引起复合物的解离，这种解离的性质表明蛋白质与膜的平衡结合。然而，当囊泡中PS含量超过20%时，PKC只能部分解离。许多比较表明，即使有磷酯存在，蛋白质与膜的结合也不足以促进酶活性的发展。例如，PS在促进活性方面比其他酸性磷脂更有效，但在蛋白质结合方面并没有很大的优势。二油苷（DAG）和酚酯是PKC活性的有效激活剂，对PKC酶与磷脂囊泡的结合或结合所需的Ca2+几乎没有影响。结合只需要相等重量浓度的磷脂和蛋白质，而活性总是需要大量过量的磷脂。这些研究表明，PKC-PS-Ca2+-DAG复合物形成以外的因素对酶活性的发展至关重要。
The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+-dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g phospholipid at steric saturation. Binding was of relatively high affinity (Kd < 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity. For example, PS was much more effective in promoting activity than the other acidic phospholipids but was not greatly superior in protein binding. Diolein (DAG) and phorbol esters, potent activators of PKC activity, had little effect on the binding of the enzyme to phospholipid vesicles or on the Ca2+ requirement of binding. Binding required only equal weight concentrations of phospholipid and protein while activity always required a large excess of phospholipid. These studies suggest that factors other than formation of a PKC-PS-Ca2+-DAG complex are critical to development of enzyme activity.