Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA.
Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA.
复制标题
钙调蛋白和 Ca-EGTA 激活胰岛细胞质膜 (Ca2 Mg2 )-ATP 酶。
DOI：
10.1016/s0021-9258(18)32497-9
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
M. Mcdaniel
中科院分区：
文献类型：
--
作者：
N. Kotagal;J. Colca;M. Mcdaniel
胰岛细胞质膜含有钙刺激和镁依赖性 ATP 酶 (Ca2+ + Mg2+)-ATP 酶，需要钙调蛋白才能获得最大酶活性（Kotagal, N.、Patke, C.、Landt, M.、McDonald, J.、Colca, J.、Lacy, P. 和 McDaniel, M. (1982) FEBS Lett. 137, 249-252）。研究表明，外源添加的钙调蛋白增加了高亲和力 (Ca2+ + Mg2+)-ATP 酶的 Ca2+ 速度并降低了 Km。这些研究通常使用螯合剂乙二醇双（β-氨基乙基醚）-N,N,N',N'-四乙酸 (EGTA) 将 Ca2+ 浓度维持在亚微摩尔范围内。在这些研究过程中，意外地发现，增加培养基中 EGTA (0.1-4 mM) 和总钙的浓度，同时保持恒定的游离 Ca2+ 水平，会增加高亲和力 (Ca2+ + Mg2+)-ATP 酶的速度。通过钙敏感电极验证这些条件下的游离钙浓度。 2-4 mM EGTA 最大程度激活的 (Ca2+ + Mg2+)-ATP 酶不会受到钙调蛋白的进一步刺激，而卡调蛋白刺激随着培养基中 EGTA 浓度的降低而增加。 Ca-EGTA 对质膜富集部分的活性钙转运也有类似的增强作用。此外，Ca-EGTA 对活性钙转运以及胰岛细胞内质网 Ca2+ 刺激的 ATP 酶活性的影响可以忽略不计，而钙调蛋白不会刺激这些过程。结果表明，Ca-EGTA 的刺激可用于区分这些亚细胞细胞器的钙转运系统。此外，常规用于维持游离Ca2+水平的EGTA浓度本身可能掩盖了钙调蛋白和其他生理制剂对钙依赖性活动的影响。
Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.