PROTEIN KINASE C--IN VITRO STUDIES

蛋白激酶C(PKC)是一种关键的调节酶，被认为是 参与许多细胞过程，如细胞生长和 分化，激素释放，血小板激活，以及许多 其他活动。底物蛋白的磷酸化通常 需要钙和磷脂以及第二信使， 甘油二酯(DAG)。生物活性佛波醇酯 替代DAG。尽管目前对此进行了大量的研究 蛋白质，对其产生的物理实体知之甚少 以及各种激活剂如何发挥它们的影响。 我们发现，大豆对钙和磷脂的需求。 PKC大大超过了PKC与膜结合所需的能力 辅因子(磷脂、钙和/或DAG)需求 PKC活性的高低取决于底物的选择。在……里面 在寻找这种行为的解释时，我们观察到 底物与磷脂的相互作用是关键 蛋白激酶C的作用和所有良好的体外底物不仅 与磷脂结合但导致磷脂聚集 水泡。这些观察结果说明有必要评估所有 PKC及其底物和辅因子的不同相互作用。 这项研究将研究PKC和其他 各种状态的成分(如磷脂小泡和 单层)，并将确定事件及其更多 详细的方面对于发展激酶的活性是重要的。 将尝试鉴定非聚集化验系统。 PKC的金属离子结合性能将通过几个 技术包括直接绑定测量和 荧光法。膜对PKC的选择性 结构特征(如组成和表面曲率)将 对PKC膜相关因素的研究 结合，并在产生不可解离的膜结合的PKC。 磷酸化对蛋白质-膜组装的影响 使用两种不同的蛋白质进行评估，组蛋白和髓鞘碱性 蛋白。Ca_2、DAG或佛波酯对大鼠血管内皮细胞功能的影响 PKC-膜组装的构象和动力学研究 并将使用荧光和CD研究解离 方法：研究方法。所有的交互都将与PKC的外观进行比较 帮助定义酶-底物的这些方面的活性- 膜-辅因子复合体对肿瘤的发展至关重要 激活酶活性。定义体外特性将改善我们的 了解这种酶在体内的靶点是什么 已确认身份。

Protein kinase C (PKC) is a key regulatory enzyme believed to be involved in many cellular processes such as cell growth and differentation, hormone release, platelet activation, and many other events. Phosphorylation of substrate proteins usually requires Ca+2 and phospholipid as well as the second messenger, diacylglycerol (DAG). Biologically active phorbol esters substitute for DAG. Despite abundant current studies on this protein, little is known about the physical entity that generates kinase activity and how various activators exert their influence. We have found that the Ca+2 and the phospholipid requirements of PKC greatly exceed those needed for PKC-membrane binding and that the cofactor (phospholipid, Ca+2, and/or DAG) requirements of PKC activation are dependent on the choice of substrate. In searching for an explanation for this behavior, we observed the interaction of substrates with the phospholipid was a critical aspect of PKC action and all good in vito substrates not only bound to phospholipid but caused aggregation of phospholipid vesicles. These observations illustrate the need to evaluate all of the different interactions of PKC, its substrates and cofactors. This study will investigate the interactions of PKC and other components in various states (e.g. phospholipid vesicles and monolayers) and will determine how the events and their more detailed aspects are important to develoment of kinase activity. Identification of nonaggregated assay systems will be attempted. Metal ion binding properties of PKC will be examined by several techniques including direct binding measurements and fluorescence methods. Selectivity of PKC for membrane structural features (e.g. composition and surface curvature) will be studied to determine the factors involved in PKC-membrane binding and in generating nondissociable membrane-bound PKC. The effect of phosphorylation on protein-membrane assembly will be assessed using two different proteins, histones and myelin basic protein. The effect of Ca+2, DAG or phorbol esters on the conformation and on the dynamics of PKC-membrane assembly and dissociation will be investigated using fluorescence and CD methods. All interactions will be compared to appearance of PKC activity to help define those aspects of the enzyme-substrate- membrane-cofactor complex that are critical to development of kinase activity. Defining the in vitro properties will improve our understanding of how in vivo targets for this enzyme are identified.