INTERACTION OF CALMODULIN WITH PHOSPHODIESTERASE AND OTHER BINDING PROTEINS

钙调蛋白与磷酸二酯酶和其他结合蛋白的相互作用

• 批准号: 3966533
• 负责人: R L KINCAID
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3966533
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; brain; brain mapping; calcium; enzyme induction /repression; enzyme linked immunosorbent assay; enzyme structure; fluorescence polarization; neurons; proteolysis; synapses

A poorly hydrolyzed substrate, N6-etheno cyclic AMP, was used to assay high concentrations (0.2 - 0.6 MuM) of cyclic nucleotide phosphodiesterase (PDE) permitting direct comparison of activity with changes in physical properties of the enzyme. Although the interaction constant for this substrate (2-3mM) was 100-fold higher than that for cAMP, the regulatory properties of the enzyme were comparable (e.g., Ka for Mg2+, Ki for spermine, degree of stimulation by calmodulin (CaM). When the Ca2+-dependence of enzyme activation was compared with that for interaction with dansyl-calmodulin (D-CaM) using identical experimental samples, less Ca2+ was required for interaction than for stimulation of activity; this suggested sequential steps in the mechanism of PDE activation by CaM. Immunocytochemical studies in rat brain indicated that specific changes in the distribution of PDE in cerebellum occurred after pharmacologic lesions of the inferior olivary nucleus, while that of calcineurin (CN) did not. Since this treatment affects excitatory innervation of Purkinje cells, it is possible that such input pathways may modulate, transynaptically, the local expression of PDE. Using overlay procedures, CN has been identified as the predominant CaM-binding protein in isolated spleen cells and cultured PC-12 cells; smaller amounts of cytoskeletal CaM-binding proteins (caldesmon, spectrin) have also been found. In some instances, there were changes in the amounts of these proteins with differentiation, suggesting a role for Ca2+-dependent protein dephosphorylation and/or cytoskeletal modification during cellular activation. Expression vector immunoscreening procedures were optimized to permit isolation of putative cDNA clones for PDE and CN using a lambda GT-aa rat brain library. Lysogens of these clones were produced and the fusion proteins analyzed for immunoreactivity against affinity-purified CN and PDE antibodies, and against monoclonal anti-beta galactosidase antibody.

水解不良的底物N6-乙烯环状AMP用于测定高 环核苷酸磷酸二酯酶（PDE）的浓度（0.2-0.6 MUM） 允许将活动与物理变化进行直接比较 酶的特性。 虽然相互作用常数 底物（2-3mm）比营地高100倍 酶的性质是可比的（例如，mg2+的ka，ki，ki for 精子，钙调蛋白（CAM）的刺激程度。 什么时候 将酶激活的Ca2+依赖性与相互作用进行了比较 使用相同的实验样品与丹氰基 - 钙统（D-CAM），较少 相互作用比刺激活性所需的Ca2+。这 CAM激活PDE激活机理的顺序步骤。 大鼠脑中的免疫细胞化学研究表明 小脑中PDE的分布发生在药物病变后 下橄榄核，而钙调蛋白（CN）的核则没有。 由于这种治疗会影响Purkinje细胞的兴奋性神经，因此 这种输入途径可能会跨突触地调节 PDE的局部表达。 使用覆盖过程，已经确定了CN 作为分离的脾细胞和 培养的PC-12细胞；较少量的细胞骨架凸轮结合蛋白 （Caldesmon，Spectrin）也发现了。 在某些情况下，有 这些蛋白质的量的变化与分化，表明 Ca2+依赖性蛋白质去磷酸化和/或细胞骨架的作用 细胞激活期间的修饰。 表达载体免疫镜 优化程序以允许分离推定的cDNA克隆 PDE和CN使用Lambda GT-AA大鼠脑文库。 这些的蛋白质 产生克隆，并分析了融合蛋白的免疫反应性 反对亲和纯化的CN和PDE抗体，并抗单克隆 抗β半乳糖苷酶抗体。