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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
井坂, 一真;鈴木, 邦明;南川, 元;吉村, 善隆
Permalink : http://hdl.handle.net/2115/68808
KEYWORDS : Ca-ATPase;骨芽細胞様細胞;カルモジュリン;Ca-ATPase;osteoblastic cell;calmodulin
Abstract
形態学的な研究から，硬組織形成部位にアルカリ性至適pHのCa-ATPaseの存在が示唆されているが酵素学的な性質の報告は少ない．そこで骨芽細胞様細胞であるMC3T3-E1細胞が保持するCa-ATPase活性に関して研究を行った．細胞を石灰化時期まで培養後回収し，超音波破砕後に遠心分離操作を行って膜分画を得た．ATP加水分解により生じた無機リン酸をChifflet法で定量してATPase活性を測定し，以下の結果を得た．１．膜分画にはカルシウム（Ca）あるいはマグネシウム（Mg）により活性化されるATPaseが存在し両酵素ともthapsigarginによって阻害された．Ca存在下のATPase活性はMgによって拮抗されることと，Mg-ATPaseを阻害するazideによって阻害されないことから両酵素は別の酵素と示唆された．２．Ca-ATPase活性はCa濃度依存性に増加して1 mMの遊離Ca濃度で飽和し，50 ％活性化濃度は0.3 mMであった．３．活性はpH依存性に増加し，pH 9.1でpH 7.5のほぼ３倍の活性を示して最大となりpH 10.0までは同程度の活性を示した．４．活性はATP加水分解の過程においてリン酸化酵素を形成するP型ATPaseの阻害薬であるvanadateとエタクリン酸によっては阻害されなかった．５．活性は２価金属イオンのキレーターであるEGTAおよびEDTAにより濃度依存性に阻害されたが，ビスホスホネートによっては阻害されなかった．６．遊離Ca濃度100 nMでは， Ca-ATPase活性はほぼ検出されないが，カルモジュリンを添加すると濃度に依存して活性は増大し，50 ％活性化濃度は約6 μMであった．７．カルモジュリン非添加における活性は，カルモジュリン拮抗薬であるW7によって濃度依存性に抑制され，50 ％阻害濃度は0.3 mMであった．以上の結果は，E1細胞にはアルカリ性至適pHのP型ではないカルモジュリン依存性Ca-ATPaseが存在することを示唆する．本酵素は，形態学的に存在が示唆されるCa-ATPaseと類似しており，硬組織形成に関与する可能性がある．
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.
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.
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