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.

使用水解不良的底物 N6-乙烯环 AMP 来测定高浓度 环核苷酸磷酸二酯酶 (PDE) 浓度 (0.2 - 0.6 MuM) 允许直接比较活动与身体变化 酶的性质。 尽管相互作用常数为 底物 (2-3mM) 比 cAMP 高 100 倍，cAMP 是调节 酶的性质具有可比性（例如，Ka 代表 Mg2+，Ki 代表 Mg2+） 精胺，钙调蛋白 (CaM) 的刺激程度。 当 将酶激活的 Ca2+ 依赖性与相互作用进行比较 与丹酰钙调蛋白 (D-CaM) 一起使用相同的实验样品，更少 Ca2+ 是相互作用所必需的，而不是刺激活动所必需的；这 提出了 CaM 激活 PDE 机制的连续步骤。 大鼠脑中的免疫细胞化学研究表明， 药物损伤后小脑中 PDE 的分布 下橄榄核，而钙调神经磷酸酶（CN）则不然。 由于这种治疗会影响浦肯野细胞的兴奋性神经支配，因此 这种输入途径可能会通过突触调节 PDE 的局部表达式。 使用覆盖程序，CN 已被识别 作为分离的脾细胞中主要的 CaM 结合蛋白， 培养的PC-12细胞；较少量的细胞骨架 CaM 结合蛋白 （caldesmon、血影蛋白）也已被发现。 在某些情况下，有 这些蛋白质的数量随着分化而变化，表明 Ca2+ 依赖性蛋白质去磷酸化和/或细胞骨架的作用 细胞激活过程中的修饰。 表达载体免疫筛选 优化程序以允许分离推定的 cDNA 克隆 使用 lambda GT-aa 大鼠大脑库进行 PDE 和 CN。 这些溶源原 产生克隆并分析融合蛋白的免疫反应性 抗亲和纯化的 CN 和 PDE 抗体，以及抗单克隆抗体 抗β半乳糖苷酶抗体。