MECHANISM AND ROLE OF CALCIUM AND PHOSPHOLIPID IN HEART

本申请的总体目标涵盖分子，免疫学， 的药理学、调节和功能方面， 磷脂/钙离子依赖性蛋白激酶（PL/Ca-PK或蛋白激酶C） 系统在心中 具体目标如下：（1）. PL/Ca-PK，The 酵素 催化剂和调节剂的鉴定、表征 天然酶的结构域（80 kDa）将使用各种方法进行， 方法，这样的免疫反应性与抗血清的天然酶， ELISA或免疫印迹，以及ATP-、磷脂酰丝氨酸-或 Ca 2+结合位点。 针对纯化的抗体的多克隆和/或单克隆抗体 结构域将被开发并用于免疫细胞化学定位 的结构域，与天然酶相比，在心肌和 受药物干预或病理生理学影响的培养的肌细胞。 的 该酶的基因将使用抗 这个实验室已经开发出的天然酶 （二）、的底物 PL/Ca-PK：将进一步探索酶的底物决定簇 使用具有氨基酸的髓鞘碱性蛋白（MBP）的合成肽， 磷酸化丝氨酸-115周围的序列片段。 的 心肌肌钙蛋白I和肌钙蛋白T中的磷酸化位点以及 将确定磷酸化位点周围的氨基酸序列。 肌钙蛋白磷酸化的功能意义将通过 研究活性和Ca 2+敏感性的任何变化， 肌动球蛋白ATP酶 （三）、PL/Ca-PK系统的调节：抑制剂 肽（MBP底物肽的类似物）将进一步研究， 修饰其氨基酸残基或肽长度。 的 纯化新鉴定的心脏内源性抑制蛋白， 确定其活性位点。 具有氨基酸序列的肽 将合成活性位点周围的片段，并对其进行测试， 抑制活性 抑制肽的作用机制 （来源于MBP、肌钙蛋白和内源性抑制剂）， 与其他已知的抑制剂（如心脏毒素I）相比。 它 希望本提案能提供有关以下方面的新知识 在蛋白磷酸化水平上调节心脏功能 由这种主要的多功能蛋白激酶催化。

The overall goal of this application covers the molecular, immunological, pharmacological, regulatory and functional aspects of the phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C) system in heart. The specific aims are as follows: (1). PL/Ca-PK, The enzyme. Identification, characterization of the catalytic and regulatory domains of the native enzyme (80 kDa) will be carried out using various methods, such a immunoreactivity with antiserum to the native enzyme by ELISA or immunoblots, and labeling of the ATP-, phosphatidylserine- or Ca2+-binding sites. Poly- and/or monoclonal antibodies to the purified domains will be developed and used for the immunocytochemical localization of the domains, compared with that of the native enzyme, in myocardium and cultured myocytes influenced by drug interventions or pathophysiology. The gene for the enzyme will be cloned using the polyclonal antibodies against the native enzyme already developed in this lab. (2). Substrates for PL/Ca-PK: The substrate determinants for the enzyme will be further probed using synthetic peptides of myelin basic protein (MBP) having amino acid sequence segments around the phosphorylated serine-115. The phosphorylation sites in cardiac troponin I and troponin T as well as the amino acid sequence around the phosphorylation sites will be determined. Functional significance of troponin phosphorylation will be studied by investigating any changes in the activity and Ca2+-sensitivity of actomyosin ATPase. (3). Regulation of PL/Ca-PK system: The inhibitor peptides (analogs of MBP substrate peptides) will be further studied by modifying their amino acid residues or peptide lengths. The newly-identified cardiac endogenous inhibitor protein will be purified, and its active site(s) determined. Peptides having amino acid sequence segments around the active sites will be synthesized and tested for its inhibitory activity. The mechanisms of action of the inhibitor peptides (derived from MBP, troponin and endogenous inhibitor) will be examined, and compared with those of other known inhibitors (such as cardiotoxin I). It is hoped that the present proposal would provide new knowledge concerning regulation of cardiac function at the level of protein phosphorylation catalyzed by this major, multifunctional protein kinase.