Purification and molecular cloning of a novel acidic calmodulin binding protein from rat brain.

An acidic protein was identified among the insoluble proteins of a growth cone-enriched fraction, and this protein was purified from the Triton- and high NaCl-insoluble fraction of newborn rat brain using several column chromatographies after solubilization at an alkaline condition. The purified protein showed a Ca(2+)-dependent calmodulin binding activity. This protein showed an anomalous behavior in SDS-polyacrylamide gel electrophoresis as that observed in case of GAP-43 (neuromodulin, F1, pp46, p57, B-50) and MARCKS (p87, p80), namely shifts of apparent molecular weights under different acrylamide concentrations. Its physicochemical characteristics, such as heat stability, acidic isoelectric point, and solubility in a 2.5% perchloric acid solution, also resemble the properties of these proteins. cDNA cloning of this protein showed that the NH2-terminal 50-amino acid sequence was almost identical to CAP-23, a previously reported chicken protein of unknown function. Since the COOH-terminal half-regions of these proteins are less similar, the entire sequences of these proteins have 65% homology (52% identity), suggesting that these proteins belong to a family. Immunoblotting of several tissue extracts using a monoclonal antibody against this protein showed its specific expression in brain.