MOLECULAR REGULATION OF CALMODULIN-DEPENDENT PHOSPHODIESTERASE AND PHOSPHATASE

钙调蛋白（CaM）调控环的推定克隆

Putative clones for the calmodulin (CaM)-regulated cyclic nucleotide phosphodiesterase (PDE) and protein phosphatase, calcineurin (CN), have been isolated from lambda gt-11 brain libraries and are being characterized. On such clone for PDE, selected by expression vector immunoscreening, contains an EcoRI insert of 135 bp, coding for the fusion protein domain. Because of its small size, it has been necessary to label the insert for use as probe in obtaining larger cDNAs. A rapid, non- radioactive method for screening phage libraries by placque hybridization was developed for this purpose. Immunocytochemical studies in rat brain indicate that, during development, PDE immunoreactivity appears to be correlated with the migration of certain neuronal populations. This suggests that the commitment to express PDE precedes the final positioning of the neurons (i.e., establishment of stabilized synaptic contact). Biochemical studies of CaM-dependent PDE in mouse testis have shown a novel isozyme that displays higher affinity for cAMP (2 MuM vs. 40 MuM), and is somewhat larger than the brain enzyme (66 kDa vs. 60 kDa). Since this PDE is intensely immunoreactive with antibody prepared against the brain enzyme, it suggest that they share common epitopes and perhaps similar structural genes. A novel 65 kDa CaM-binding protein (CaM-BP) was observed in murine thymocytes in contrast to that seen in other lymphocyte fractions, (B-,T-cells) where CN was the predominant BP. However, comparison of the proteolytic fragments indicate that the thymus-specific protein may be a precursor form of the phosphatase, consistent with "processing" of this enzyme during lymphocyte development (i.e., into T-cells).