Ichikawa Emiko, Tsurusaki Yoshinori, Yamaguchi Masayoshi
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Int J Mol Med. 2004 Feb;13(2):289-93.
The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.
利用正常（野生型）和瑞谷钙素转基因（TG）大鼠，研究了细胞内信号通路中的调节蛋白瑞谷钙素在心肌细胞质中对蛋白磷酸酶活性调节的作用。在含有磷酸酪氨酸、磷酸丝氨酸和磷酸苏氨酸的反应混合物中，测定了含有细胞质蛋白的蛋白磷酸酶活性。在酶反应混合物中加入氯化钙（10和20微摩尔），导致蛋白磷酸酶对三种磷酸氨基酸的活性显著增加。三氟拉嗪（10和20微摩尔），一种钙调蛋白拮抗剂，完全抑制了钙（10微摩尔）添加后蛋白磷酸酶对三种磷酸氨基酸活性的增加。此外，加入钙调蛋白（2.5或5微克/毫升）显著增强了钙（10微摩尔）对磷酸丝氨酸和磷酸苏氨酸的酶活性增加。在存在磷酸酪氨酸的情况下未观察到这种增强作用。在不添加Ca2+且存在乙二醇双（2-氨基乙醚）N,N,N',N'-四乙酸（EGTA；1毫摩尔）的情况下，瑞谷钙素（10-9和10-8摩尔）显著抑制蛋白磷酸酶对三种磷酸氨基酸的活性。在存在氯化钙（10微摩尔）的情况下，也观察到了瑞谷钙素（10-10 - 10-8摩尔）的抑制作用。蛋白质印迹分析表明，与野生型雌性大鼠相比，瑞谷钙素TG雌性大鼠心脏细胞质中瑞谷钙素蛋白表达显著。TG大鼠心脏细胞质中蛋白磷酸酶对三种磷酸氨基酸的活性显著降低。在TG大鼠心脏细胞质中未观察到添加钙（10微摩尔）对蛋白磷酸酶对三种磷酸氨基酸活性的增强作用。本研究表明，内源性瑞谷钙素在大鼠心脏细胞质中蛋白磷酸酶活性的调节中起抑制作用。
