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Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity

机译:花生四烯酸和二酰基甘油在体外协同激活蛋白激酶C:产生稳定的膜结合,辅因子非依赖性蛋白激酶C活性状态

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摘要

The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium dictation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.
机译:本研究检查了磷脂囊泡中花生四烯酸和二酰基甘油对PKC的协同活化作用,并证明了这种活化剂的结合导致了该酶的组成型活性,磷脂结合形式的形成。 PKC的活化几乎完全依赖钙,而仅含有二油酰基甘油的囊泡。相反,当囊泡同时含有二酰基甘油和游离花生四烯酸时,观察到相当大的钙非依赖性活性。酶活性与含二酰基甘油的囊泡的高亲和力关联是钙依赖性且可逆的。然而,在没有钙的情况下,向含有二酰基甘油的囊泡中添加花生四烯酸会导致不可逆的PKC结合。免疫印迹分析表明,钙依赖性结合不是同工酶特异性的。在没有钙的情况下与囊泡结合的与囊泡相关的PKC的活性主要是钙依赖性的。另一方面,当在钙存在下进行囊泡结合酶的结合和分离时,随后的活性几乎完全抵抗钙命令。当洗涤剂被提取并与磷脂囊泡重组时,这种与囊泡相关的酶形式保持了显着的“组成”活性(在不存在二酰基甘油和钙的情况下具有活性)。来自该体外系统的数据为PKC的生理调节模型提供了基础,其中花生四烯酸酯和二酰基甘油的联合作用促进了与膜紧密相关的内在活性形式的PKC的稳定形成。

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