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.
Maekawa
S
S
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Maekawa
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M
Hattori
S
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Nakamura
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017630:Connexins; D003598:Cytoskeletal Proteins / Q000737:chemistry; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry; D051381:Rats; D017386:Sequence Homology, Amino Acid
D002148:Calmodulin-Binding Proteins; D017630:Connexins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C066853:Basp1 protein, rat; D004247:DNA