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Title: 骨芽細胞様細胞(MC3T3-E1)のthapsigargin感受性Ca依存ATPaseの分離
Other Titles: Separation of thapsigargin-sensitive Ca-dependent adenosinetriphosphatase in osteoblastic cells (MC3T3-E1)
Authors: 工藤, 智也1 Browse this author
出山, 義昭2 Browse this author
吉村, 善隆3 Browse this author →KAKEN DB
鈴木, 邦明4 Browse this author →KAKEN DB
山崎, 裕5 Browse this author →KAKEN DB
Authors(alt): Kudo, Tomonari1
Deyama, Yoshiaki2
Yoshimura, Yoshitaka3
Suzuki, Kuniaki4
Yamazaki, Yutaka5
Keywords: 骨芽細胞様細胞
Ca-dependent ATPase
osteoblastic cell
Issue Date: 15-Sep-2021
Publisher: 北海道歯学会
Journal Title: 北海道歯学雑誌
Volume: 42
Start Page: 28
End Page: 35
Abstract: 骨芽細胞にはCaで活性化されるATPase(Ca-ATPase)が存在し石灰化との関連が推測されるが報告は少ない.そこで,骨芽細胞様細胞(MC3T3-E1)のCa-ATPaseの性質を調べた.E1細胞を培養後,細胞破砕物を作成したのち,ミクロソーム分画とした.さらにドデシル硫酸ナトリウム(SDS)を添加して可溶化した後,グリセロールの密度勾配遠心にかけてATPase活性のある分画を得た.細胞破砕物にはCa,Mg-ATPase以外に,pH 9.6( Ca-P1)とpH10.8(Ca-P2)で活性が高いCa-ATPase及びpH 9.6(Mg-P1)が主でpH11.1(Mg-P2)にわずかな活性を示すMg-ATPase活性が検出された.Mg-P1の最大活性はCa-P1及びCa-P2の約4.5倍であった.これらの活性はSERCAの特異的阻害剤とされるthapsigarginによって阻害されたが,P型ATPaseを阻害するvanadateによる阻害は認められなかった.また2価金属のキレーターであるEDTA及びEGTAは濃度依存性に両ATPase活性を阻害した.ミクロソーム分画ではCa-P1が顕著に減少し,Ca-P2の割合が増加した.グリセロール密度勾配遠心後には,Mg-P2とその約3倍のCa-P2活性が検出された.両活性はthapsigarginにより阻害された.両活性の石灰化への関与を調べるために,25及び50μMのthapsigargin存在下でのE1細胞の培養を行ったが,thapsigarginのアポトーシス誘導作用のため石灰化の検出まで細胞を培養することはできなかった.本研究ではthapsigarginにより阻害されP型ではないアルカリ性至適pH のCa-ATPaseがE1細胞に存在することを示し,その性質の検討が可能な程度まで分離を行うことができた.
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
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