Separation of thapsigargin-sensitive Ca-dependent adenosinetriphosphatase in osteoblastic cells (MC3T3-E1)

Adenosinetriphosphatase (ATPase) activated by calcium ions (Ca-ATPase) exists and plays a role in bone mineralization; however, not enough research has been conducted on it. Therefore, this report studied the properties of Ca-ATPase in osteoblastic cells (MC3T3E-1). Osteoblastic cells were cultured and harvested at 4 weeks after confluence. The cells were homogenized through ultrasonication and separated into the microsome and cytoplasm by centrifugation. Further, they were solubilized using sodium dodecyl sulfate and separated into fractions, including Ca-ATPase activity on the density gradient of glycerol. In addition, the cell homogenate detected high Ca-ATPase activity at a pH of 9.6 (Ca-P1) and 10.8 (Ca-P2), and Mg-ATPase activity at a pH of 9.6 (Mg-P1). Low Mg-ATPase activity at a pH of 11.1 (Mg-P2) was also detected. The maximum activity of Mg-P1 was 4.5 times higher than that of Ca-P1 and Ca-P2. These activities of ATPase were inhibited by thapsigargin, the specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, but not by vanadate, an inhibitor of P-type ATPase. Ethylene glycol-bis (β-aminoethyl ether) -N,N,N′,N′-tetraacetic acid and ethylenediaminetetraacetic acid, chelators of divalent metallic ions, inhibited both ATPases concentration-dependently. In the microsome fractions, the activity of Ca-P1 decreased remarkably. The activities of Ca-P2, Mg-P1, and Mg-P2 remained the same, but the ratio of Ca-P2 increased. Mg-ATPase was inhibited by azide, an inhibitor of mitochondrial ATPase, but Ca-ATPase was not inhibited. After centrifugation of the density gradient of glycerol, the activity of Mg-P1 decreased remarkably, but the activities of Mg-P2 and Ca-P2 were maintained. The activity of Ca-P2 was found to be thrice higher than that of Mg-P2. Both ATPases were inhibited by thapsigargin. To investigate the role of both ATPases in mineralization, MC3T3-E1 cells were cultured with 25 and 50 μM thapsigargin. Three days after culture, the surface layers of cells separated, and the number of cells decreased. As thapsigargin strongly induced cell apoptosis, we could not culture the cell until mineralization was detected. These results suggested that the E-1 cell has Ca-ATPase with optimal pH at the alkaline side which is inhibited by thapsigargin but not by P-type ATPase inhibitor. In addition, we could separate Ca-ATPase to the extent required for use in its characterization