骨芽細胞様細胞（MC3T3-E1）のカルモジュリン依存性Ca-ATPase活性の性質

Ca-ATPase

北海道歯学会

北海道歯学雑誌

Morphological studies suggest the presence of Ca-ATPase at alkaline pH optimum at the site of hard tissue formation, but there are few reports on enzymological properties. Therefore, we investigated Ca-ATPase activity using clonal osteoblastic cells (MC3T3-E1). The cells were cultured until calcification. The cells were then ultrasonically crushed. Further, they were centrifuged to obtain a membrane fraction. The ATPase activity was measured by measuring inorganic phosphate produced by ATP hydrolysis by the Chifflet method. The following results were obtained : 1. Ca-ATPase and Mg-ATPase exist in membrane fractionation. They are inhibited by thapsigargin. Ca-ATPase activity is inhibited by Mg. Mg-ATPase is inhibited by azide, but Ca -ATPase was not inhibited. Both are different enzymes. 2. Ca-ATPase activity increased with Ca concentration dependence, saturated at 1 mM free Ca concentration, and 50% activation was 0.3 mM. 3. Ca-ATPase activity increased in pH dependence and showed maximum activity at pH 9.1. It is nearly three times that of pH 7.5. It is the same value up to pH 10.0. 4. Ca-ATPase activity is not inhibited by vanadate, an inhibitor of P-type ATPase. Its activity is not inhibited by ethacrynic acid, an inhibitor of Mg-ATPase. 5. Ca-ATPase activity is inhibited in a concentration-dependent manner by EGTA and EDTA of bivalent metal chelators. Its activity is not inhibited by bisphosphonates. 6. At free Ca concentration of 100 nM, Ca-ATPase activity in the absence of calmodulin was not detected. Ca-ATPase activity increases with the addition of calmodulin depending on the concentration, and the 50 % activation concentration is about 6 μM. 7. Ca-ATPase activity is inhibited in a concentration-dependent manner by W7 which is a calmodulin antagonist when calmodulin is not added. The 50 % inhibitory concentration is 0.3 mM. The above results suggest that Ca-ATPase in MC3T3-E1 cells is calmodulin-dependent, which is optimal at alkaline pH and not P-type. This enzyme may be identical to Ca-ATPase which is morphologically suggested to be present, and it may be involved in hard tissue formation.