PROTEIN KINASE C--IN VITRO STUDIES

此应用程序用于继续支持旨在 蛋白激酶C(PKC)与其相互作用的研究 辅因子，并确定其被激活的机制。PKC 是一种重要的细胞内调节蛋白，也是 亲和力受体为肿瘤促进剂佛波酯。活动 蛋白激酶C的活性依赖于钙和磷脂。在低钙条件下 浓度时，佛波酯和/或佛波醇酯也能刺激活性。 甘油二酯。PKC以钙依赖的方式与膜结合，并 它也是一种新发现的钙反应类别的成员 只有在膜存在的情况下才能结合钙的蛋白质。PKC表格 两种膜结合状态，一种是可逆的和钙依赖的，而 另一种具有完整膜蛋白的性质。形成 后者受佛波醇酯的刺激。这项研究建议 继续阐明激活和相互作用的机制 与这些辅因子有关的PKC的表达。磷脂酰乙醇胺的作用， 细胞内主要的中性磷脂--PKC及其各种 我们将研究它们的特性。初步结果显示人们非常倾向于 对于这种磷脂来说，磷脂酰胆碱是主要的中性 细胞表面有磷脂。我们计划记录并解释 这种差异的基础是使用酶活性和 蛋白-膜结合。其他目标包括确定 促进整合体形成的条件和磷脂 膜结合形式的PKC。这种形式的蛋白质将被检测到 它的辅因子非依赖性活性及其与磷脂的洗脱 研究了EGTA存在下的凝胶过滤层析。一个主要目标 将决定佛波醇酯或佛波醇酯 二酰甘油在可逆膜结合状态下激活PKC。 蛋白质-膜的相互作用将用荧光或光来研究。 散射技术和活性将由蛋白质来衡量 与放射性三磷酸腺苷的磷酸化。荧光光谱和CD光谱 与磷脂相互作用引起的PKC的变化， 将通过测量佛波酯和钙来确定 与这些相互作用相关的蛋白质结构变化。另一个 已分离出大量结合钙和钙的蛋白质 膜以类似于PKC的方式。第一个目标将是 确定他们的身份，以及他们是否属于 蛋白质。这些不同蛋白质对膜特性的影响 将被研究以确定它们是否在体内产生膜通透性 它们的可逆或不可逆膜结合形式。扩展 目标是继续检查各种PKC激活剂的机制 和抑制剂，并比较这些不同性质的不同 PKC的异构体。这些结果应该有助于揭开 佛波酯效应与某些秒的作用机制 信使。

This application is for continuation of support for a project designed to characterize the interaction of Protein Kinase C (PKC) with its cofactors, and to determine the mechanisms by which it is activated. PKC is an important intracellular regulatory protein that is also the high affinity receptor for the tumor promotors, phorbol esters. The activity of PKC is calcium and phospholipid- dependent. At low calcium concentrations, the activity is also stimulated by phorbol esters and/or diacylglycerol. PKC binds to membranes in a calcium-dependent manner and it is also a member of a newly identified category of calcium response proteins that bind calcium only in the presence of membranes. PKC forms two membrane-bound states, one is reversible and calcium-dependent while the other has properties of an integral membrane protein. Formation of the latter is stimulated by phorbol esters. This study proposes to continue the elucidation of the mechanism of activation and interaction of PKC with these cofactors. The effect of phosphatidyl- ethanolamine, the major intracellular neutral phospholipid, on PKC and its various properties will be studied. Preliminary results show a great preference for this phospholipid over phosphatidyl-choline, the major neutral phospholipid on the cell surface. We plan to document and explain the basis for this difference using assays for both enzyme activity and protein-membrane binding. Other objectives include determination of the conditions and phospholipids that enhance formation of the integral membrane-bound form of PKC. This form of the protein will be detected by its cofactor- independent activity and by its elution with phospholipids on gel filtration chromatography in the presence of EGTA. A major goal will be to determine the mechanism by which Phorbol esters or diacylglycerol activate PKC in its reversible membrane-bound state. Protein-membrane interaction will be studied by fluorescence or light scattering techniques and activity will be measured by protein phosphorylation with radioactive ATP. The fluorescence and CD spectral changes in PKC that are induced by interaction with phospholipids, phorbol esters and calcium will be measured to determine the extent of protein structural change associated with these interactions. Another abundant group of proteins has been isolated that bind calcium and membranes in a manner similar to PKC. The first objective will be to determine their identity and if they are members of the annexin family of proteins. The effects of these various proteins on membrane properties will be studied to determine if they produce membrane permeability in either their reversible or irreversible membrane-bound forms. Extended goals are to continue to examine the mechanisms of various PKC activators and inhibitors, and to compare these various properties for the different isoforms of PKC. These results should help unravel the basis for the phorbol ester effect and the mechanism of action of some second messengers.