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.
