Takamatsu K, Kitamura K, Noguchi T
Department of Physiology, Toho University School of Medicine, Tokyo, Japan.
Biochem Biophys Res Commun. 1992 Feb 28;183(1):245-51. doi: 10.1016/0006-291x(92)91635-4.
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.
通过免疫印迹分析发现，大鼠脑中存在一种分子量为23,000的蛋白质（P23k），它与恢复蛋白明显不同，并用针对恢复蛋白NH2末端产生的抗血清进行标记。针对恢复蛋白COOH末端产生的抗血清无法检测到P23k。用45Ca进行的印迹显示P23k结合Ca2+。这种钙蛋白通过Ca(2+)依赖性疏水相互作用和离子交换色谱进一步纯化。在SDS聚丙烯酰胺凝胶电泳中，在存在10 microM Ca2+的情况下，P23k的表观分子量为21,000，在不存在Ca2+（0.1 mM EGTA）的情况下为23,000。P23k的等电点为5.6。Ca(2+)结合分析表明，P23k每摩尔蛋白质结合2摩尔Ca2+，有两个结合位点，解离常数分别为13 microM和0.2 microM。纯化的P23k以Ca(2+)依赖性方式与小脑、大脑和视网膜的粗膜部分结合。对P23k蛋白水解片段的部分氨基酸序列分析揭示了P23k与恢复蛋白之间的序列同源性。这些结果表明，P23k可能通过与其膜上的靶标形成复合物而作为Ca(2+)敏感调节剂发挥作用。
