MECHANISM AND ROLE OF CALCIUM AND PHOSPHOLIPID IN HEART

这项应用的总体目标包括分子、免疫学、 在药理、调节和功能方面 磷脂/钙依赖的蛋白激酶(PL/Ca-PK，或蛋白激酶C) 内心的系统。具体目标如下：(1)。PL/Ca-PK、 酵素。催化剂和调节剂的鉴定和表征 天然酶(80 KDa)的结构域将使用各种不同的 方法，这种与天然酶抗血清的免疫反应是通过 ELISA法或免疫印迹法，以及ATP、磷脂酰丝氨酸或 Ca2+结合部位。纯化后的多克隆抗体和/或单抗 结构域将被开发并用于免疫细胞化学定位 与天然酶相比，在心肌和 培养的心肌细胞受药物干预或病理生理学的影响。这个 将使用针对该酶的多克隆抗体来克隆该酶的基因 在这个实验室里已经研发出的天然酶。(2)。基材用于 PL/Ca-PK：酶的底物决定因素有待进一步探讨 使用含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。这个 心肌肌钙蛋白I和肌钙蛋白T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 研究酶活性和钙敏感性的任何变化 肌动球蛋白ATPase。(3)。磷脂酰肌醇/钙-磷酸酶系统的调节：抑制剂 多肽(MBP底物多肽的类似物)将通过 修饰它们的氨基酸残基或多肽长度。这个 新发现的心脏内源性抑制蛋白将被提纯，并 其活性部位(S)确定。具有氨基酸序列的多肽 活性部位周围的片段将被合成并测试其 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物)将被检测，以及 与其他已知的抑制剂(如心脏毒素I)相比。它 希望本提案将提供有关以下方面的新知识 蛋白质磷酸化水平对心功能的调节 由这种主要的、多功能的蛋白激酶催化。

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.