Characterization and Biosynthesis of Cyclic‐AMP‐Binding Proteins in the Rat Central Nervous System

Abstract: Cyclic‐AMP‐binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8‐azido‐[³²P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic‐AMP‐binding proteins. Labeled proteins were then analyzed by two‐dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic‐AMP‐binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the β‐tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic‐AMP‐binding proteins adjacent to the β‐tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic‐AMP‐binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic‐AMP‐binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffmity‐labeled in cytosol and membrane fractions. A translation product at 54K 5.3 was not detected, and, therefore, the possibility exists that this protein may result from posttranslational modification of another protein. The translation product at 52K 5.3 was precipitated with antibodies against the brain specific type II regulatory subunit of cyclic‐AMP‐dependent protein kinase.