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Mg,Ca及びZnのヒトアルカリ性ホスファターゼ活性に対する作用

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Title: Mg,Ca及びZnのヒトアルカリ性ホスファターゼ活性に対する作用
Other Titles: Effects of Mg, Ca, and Zn on human alkaline phosphatase activity
Authors: 半谷, 純一1 Browse this author
鈴木, 邦明2 Browse this author →KAKEN DB
吉村, 善隆3 Browse this author →KAKEN DB
南川, 元4 Browse this author →KAKEN DB
兼平, 孝5 Browse this author →KAKEN DB
本多, 丘人6 Browse this author →KAKEN DB
Authors(alt): Hanya, Junichi1
Suzuki, Kuniaki2
Yoshimura, Yoshitaka3
Minamikawa, Hajime4
Kanehira, Takashi5
Honda, Okahito6
Keywords: アルカリ性ホスファターゼ
ナトリウムピロリン酸
マグネシウム
カルシウム
亜鉛
alkaline phosphatase
Ca
Mg
sodium pyrophosphate
Zn
Issue Date: 15-Sep-2022
Publisher: 北海道歯学会
Journal Title: 北海道歯学雑誌
Volume: 43
Start Page: 9
End Page: 18
Abstract: ヒトには遺伝子型の異なる4 種に加えて糖鎖による修飾が異なる多数のアルカリ性ホスファターゼ(ALP)アイソザイムが存在するが,骨型ALPのピロリン酸及び小腸型ALPのピリドキサルリン酸を除いて生理的な基質と機能に関しては不明な点が多く,また活性の至適pHがアルカリ性である意味も不明である. ヒト胎盤型ALPの結晶構造解析の結果からヒトのALPには1 サブユニットあたり1 Mg及びCaと2 Znの結合部位の存在が示されたが,ALP活性における各イオンの機能については報告が少ない.そこで,ヒトALPアイソザイムに対する各イオンの作用を比較することにより,アイソザイムによる活性の至適pH及び基質への親和性の相違を明らかにすることを目的に研究を行った.市販のヒト骨型,肝臓型,胎盤型及び小腸型ALPを使用した.基質にはパラニトロフェニルリン酸(pNPP),Na-PPi及びATPを使用し以下の結果を得た.1)pNPPを基質として,4 種類のALP活性はMg,Ca及びZn濃度依存性に増加し,50%活性化濃度はALPの種類にはよらず,Zn,Ca,Mgの順に増加した.Znは低濃度では活性を促進したが,高濃度では逆に抑制し, 2個のZn結合部位の性質は異なることを示唆した.また,各ALP活性の至適pHはALP,基質及びイオンの種類により変化した.2)pNPP 及びNa-PPiを基質として,骨型ALP活性はMg,Ca及びZn濃度依存性に増加したが,濃度依存曲線は基質によって異なった.3)MgとCaあるいはMgとZnを組み合わせて添加することにより骨型ALP活性は相加的に増加した.4)イオン及び基質の種類によらず,骨型ALP活性は阻害剤であるlevamisoleあるいはtetramisoleによって濃度依存的に阻害された.以上の結果はin vitroでは4 種類のALPには Mg,Ca及びZn結合部位が存在し独立してALP活性の促進が可能であること,活性の至適pH及び基質に対する親和性は存在するイオンによって変化しうることを示唆した.In vivoでもALPの生体内での基質は存在する2 価金属イオンの種類と周囲のpHによって変化する可能性がある.
Objectives: One Mg, one Ca, and two Zn binding sites per subunit of the human placenta type alkaline phosphatase (ALP) were detected by analyzing its crystalline structure. It was an ticipated that other human ALPs also contain these ion binding sites, as there are few reports about the function of each of these ions on ALP activity. We examined the affinity of ALP isozymes for Mg, Ca, and Zn. Moreover, we also examined the effects of Mg, Ca, and Zn on the optimum pH for ALP activity and substrate specificity. Methods: We utilized commercial human bone, liver, placenta, an d small intestine type ALP, p-nitrophenyl phosphate (pNPP), and Na-PPi as substrates, and assayed the Pi released from the substrates using the Chifflet method. Results and discussion: 1) The ALP activity of all four isozyme s increased in Mg, Ca, and Zn concentration-dependent manner with pNPP as the substrate. Remarkably, the 50% activated concentrations (K0.5) of each ion did not depend on the type of ALP and increased as the concentration of Zn, Ca, a nd Mg increased. Zn promoted the activity with a low concentration, but decreased it with a higher concentration, suggesting that the first Zn binding site stimulates activity but the second one inhibits it. The optimum pH for ALP activity varied according to the ion present. We observed that Zn, Ca, and Mg binding sites are present in all ALP. 2) The concentration dependency of Mg, Ca, and Zn on the bone ALP activity was examined using pNPP and Na-PPi as substrates. Its activity was promoted by Mg, Ca, and Zn in a concentration dependent manner, but the concentration dependency curve was different between pNPP and Na-PPi, suggesting that the affinity for Mg, Ca, and Zn depends on the substrate. 3) The activity increased remarkably when divalent cations were present in combination with Mg, Ca, and Z n, suggesting that each ion promoted ALP activity independently. 4) The ALP activity in the presence of each ion was inhibited in a concentration-dependent manner by specific ALP inhibitors such as levamisole and tetramisole. Moreover, the inhibition did not depend on the substrate used. 5) Substrate concentration-dependency of ALP activity was measured in the presence of Mg, Ca, and Zn. The K0.5 values were 0.73 mM, 0.57 mM, and 0.53 mM for pNPP with Mg, Ca, and Zn, respectively. The K 0.5 values were 1.8 mM, 0.35 mM, and 0.15 mM for Na-PPi with Mg, Ca, and Zn, respectively. The affinity for the substrate of ALP varied according to the type of substrate but not to the type of ion. Conclusion: Mg, Ca, and Zn possibly promote ALP activity independently. Our results suggest that the affinity for the substrate and the optimum pH could change depending on the ions present. It is possible that the function of ALP changes when divalent cations and a certain substrate is present.
Type: article
URI: http://hdl.handle.net/2115/86831
Appears in Collections:北海道歯学雑誌 > 第43巻

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