INTERACTION OF CALMODULIN WITH PHOSPHODIESTERASE AND OTHER BINDING PROTEINS

丹磺酰-钙调蛋白与脑相互作用的钙依赖性 比较了钙调素依赖性磷酸酶钙调神经磷酸酶(CN)和钙调素依赖的钙调神经磷酸酶 使用相同的实验样品进行酶激活。复合体形成 (荧光偏振)没有协作性(K1/2等于5-6微米 Ca~(2+))，而磷酸酶的激活是高度合作和必需的 相当于高3倍的钙离子浓度。也得到了类似的结果 对于环核苷酸磷酸二酯酶(PDE)使用替代 底物为N6-亚乙基环状AMP。这些结果与一个 钙离子依赖的相互作用和酶刺激的时序机制 其中，Ca~(2+)的扩散对激活-失活是限速的。 兔(抗PDE)和山羊(抗CN)免疫球蛋白的亲和纯化 通过ELISA法显示不到1%的交叉反应，并且没有反应 与其他钙调蛋白结合蛋白(CaM-Bps)、PDE或其他脑 蛋白质的免疫印迹分析。CN和PDE在脾组织中的存在 用这些抗体检测细胞群和PC-12细胞； 此外，用生物素标记的CaM显示所有细胞内的CaM-Bps (BioCaM)，一种为覆盖程序开发的新衍生品。该地区 并观察PDE和CN在大鼠脑内的亚细胞分布。 CN和PDE均为神经细胞；胶质细胞和其他非神经细胞 组织无反应。CN似乎无处不在地分布，并被 在突触前和突触后结构中均有发现；它表现出明显的 免疫反应呈点状，特别是沿细胞膜(突触)。pde 定位于一小部分神经的树突和胞体中 接受汇聚输入(小脑浦肯野细胞、皮质和 海马旁中层细胞)；它完全出现在突触后， 均匀分布于细胞质中。这些数据表明 CaM-Bps在中枢神经系统中的细胞特异性表达 提示CN在突触传递中起普遍作用，而PDE可能 参与选定神经元中神经输入的整合。

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.