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: 4694488
• 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/4694488
• 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

The Ca2+-dependence of interaction of dansyl-calmodulin (CaM) with brain CaM-dependent phosphatase, calcineurin (CN), was compared with that of enzyme activation using identical experimental sample. Complex formation (fluorescence polarization) showed no cooperativity (K1/2 equal to 5-6 MuM Ca2+) while phosphatase activation was highly cooperative and required equal to 3 times higher Ca2+ concentrations. Similar results were obtained for cyclic-nucleotide phosphodiesterase (PDE) using an alternative substrate, N6-etheno cyclic AMP. These results are consistent with a sequential mechanism of Ca2+-dependent interaction and enzyme stimulation wherein diffusion of Ca2+ is rate-limiting for activation-deactivation. Affinity-purified IgG fractions from rabbit (anti-PDE) and goat (anti-CN) showed less than 1% cross-reactivity by ELISA procedures and did not react with other calmodulin-binding proteins (CaM-BPs), PDEs or other brain proteins using immunoblot analysis. The presence of CN and PDE in spleen cell populations and PC-12 cells was examined using these antibodies; in addition all CaM-BPs in these cells were visualized with biotinylated CaM (bioCaM), a new derivative developed for overlay procedures. The regional and subcellular distribution of PDE and CN in rat brain was investigated. Both CN and PDE were exclusively neuronal; glial and other non-neuronal tissue was unreactive. CN appeared to be ubiquitously distributed and was found in both pre- and post-synaptic structures; it exhibited distinct punctate immunoreactivity, especially along cell membranes (synapses). PDE was localized in dendrites and soma of a small subset of nerves which receive convergent input (cerebellar Purkinje cells, cortical and hippocampal byramidal cells); it appeared entirely post-synaptic and was homogenously distributed in the cytoplasm. These data demonstrate cell-specific expression of CaM-BPs in the central nervous system and suggest a general role for CN in synaptic transmission while PDE may be involved in the integration of nervous input in selected neurons.

Dansyl-Calmodulin（CAM）与脑的相互作用的Ca2+依赖性 将CAM依赖性磷酸酶，钙调神经酶（CN）与 使用相同的实验样品激活酶。 复合形成 （荧光极化）没有合作性（K1/2等于5-6 MUM Ca2+）虽然磷酸酶激活高度合作，并且需要 等于Ca2+浓度的3倍。 获得了类似的结果 使用替代方案用于环核苷酸磷酸二酯酶（PDE） 底物，N6-乙烯环状AMP。 这些结果与 Ca2+依赖性相互作用和酶刺激的顺序机理 其中Ca2+的扩散是激活二次活化的速率限制。 来自兔（抗PDE）和山羊（抗CN）的亲和纯化的IgG级分 通过ELISA程序显示出少于1％的交叉反应性，并且没有反应 与其他钙调蛋白结合蛋白（CAM-BP），PDE或其他大脑 使用免疫印迹分析的蛋白质。 脾脏中CN和PDE的存在 使用这些抗体检查细胞群和PC-12细胞。在 用生物素化CAM可视化这些细胞中的所有CAM-BP （BioCam），一种用于叠加程序的新衍生物。 区域 研究了大鼠脑中PDE和CN的亚细胞分布。 CN和PDE均仅是神经元;神经胶质和其他非神经元 组织无反应。 CN似乎无处不在分布，并且 在突触前和突触后结构中发现；它表现出独特的 点状免疫反应性，尤其是沿细胞膜（突触）。 PDE 被定位在一小部分神经中的树突和躯体中 接收收敛输入（小脑Purkinje细胞，皮质和 海马bymidal细胞）；它看起来完全是突触后的，是 均匀分布在细胞质中。 这些数据证明了 CAM-BP在中枢神经系统中的细胞特异性表达和 提出CN在突触传播中的一般作用，而PDE可能是 参与了选定神经元中神经输入的整合。