Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.
Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.
Takamatsu
K
K
Department of Physiology, Toho University School of Medicine, Tokyo, Japan.
Kitamura
K
K
Noguchi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification*; D001921:Brain / Q000276:immunology; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D051603:Recoverin; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution
D000951:Antigens, Neoplasm; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; C496693:Hpca protein, rat; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C496733:Rcvrn protein, rat; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium