MECHANISM AND ROLE OF CYCLIC GMP AND CALCIUM IN HEART

本研究的目的是研究1)钙离子的作用机制 与蛋白质磷酸化有关的离子及其磷脂的调节 和钙调蛋白，2)钙离子刺激的心肌蛋白的磷酸化， CGMP和/或cAMP及其相互关系，3)可能在 肌膜和肌浆网结合、运输和/或 钙离子、cGMP和/或cAMP依赖导致的钙离子摄取 蛋白质的磷酸化，以及4)心脏活动的可能影响 药物和心脏的生理病理状态的参数提到的 1)、2)和3)。磷脂敏感和钙调蛋白敏感的钙 离子依赖蛋白激酶将从牛中提纯到均一 心脏(和/或大脑)通过包括可控孔玻璃在内的程序， 磷脂酰丝氨酸-Affigel 102和钙调蛋白-Sepharose4B层析。 大鼠和/或豚鼠心脏内源性蛋白的磷酸化， 钙离子、cGMP和/或cAMP刺激及外源性或放射性标记 内源性(伽马-32P)三磷酸腺苷，将由十二烷基钠鉴定 硫酸盐-聚丙烯酰胺凝胶电泳法/放射自显影。隔离和 内源底物蛋白的纯化将通过吸收 (35S)有机汞亲和凝胶上的硫代磷酸化蛋白(Affigel 501)和 特别是用二硫苏糖醇洗脱。蛋白激酶的分布 和它们在心肌中的底物，以及生理病理状态的影响 关于这些参数，将通过放射免疫标记这些蛋白质进行研究 抗体/(125I)蛋白A技术在聚丙烯酰胺凝胶上的分离 然后是放射自显影。

The objectives of the research are to study 1) the mechanism of action of Ca 2 ion pertaining to protein phosphorylation, and its regulation by phospholipid and calmodulin, 2) phosphorylations of cardiac proteins stimulated by Ca 2 ion, cGMP and/or cAMP and their interrelations, 3) possible modifications in properties of sarcolemma and sarcoplasmic reticulum to bind, transport and/or uptake Ca 2 ion as results of Ca 2 ion-, cGMP-and/or cAMP-dependent phosphorylations of their proteins, and 4) possible effects of cardioactive agents and physio-pathologic states of the heart on the parameters mentioned in 1), 2) and 3). Phospholipid-sensitive and calmodulin-sensitive Ca 2 ion-dependent protein kinases will be purified to homogeneity from the bovine heart (and/or brain) by procedures including controlled-pore glass, phosphatidylserine-Affigel 102 and calmodulin-Sepharose 4B chromatographies. Phosphorylations of endogenous proteins in the rat and/or guinea pig heart, stimulated by Ca 2 ion, cGMP and/or cAMP and radiolabeled by exogenous or endogenous (gamma-32P)ATP, will be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. Isolation and purification of endogenous substrate proteins will be done by absorbing the (35S)thiophosphorylated proteins on organomercury-affinity gel (Affigel 501) and specifically eluting them with dithiothreitol. Distribution of protein kinases and their substrates in the myocardium, and effects of physiopathologic states on these parameters, will be studied by radioimmunolabeling these proteins separated on polyacrylamide gels with the antibody/(125I)protein A technique followed by autoradiography.