Purification and characterization of an Mr 87,000 protein kinase C substrate from rat brain

We (Kligman, D., and Patel, J. (1986) J. Neurochem. 47, 298-303) and others have previously identified a major protein kinase C substrate of apparent molecular weight 87,000 (Mr 87,000). To gain insight into the function of this potentially important phosphoprotein, we have undertaken its purification and characterization from rat brain. We now report a purification scheme involving heat treatment, ammonium sulfate precipitation, anion ion-exchange and reversed-phase chromatography. This procedure gave a Mr 87,000 that was homogeneous (based on silver staining), 1,600-fold enriched relative to heat-treated material and at a yield of approximately 58 micrograms/kg wet weight. We also report the amino acid composition to be high in acidic residues and in alanine and show the protein to be phosphorylated on serine residues with a stoichiometry of 2 mol of phosphate/mol of substrate. The subcellular distribution indicates Mr 87,000 is present in two forms, membrane-bound and soluble. The membrane-bound Mr 87,000 represents 45% of the total phosphoprotein content and is enriched in microsomal and synaptic membranes. Ontogenic study has revealed this protein to be developmentally regulated, with the highest concentrations of Mr 87,000 found in prenatal animals. The availability of a purification procedure should greatly facilitate further structural characterization and elucidation of the function of Mr 87,000.