Coelho-Sampaio T, Teixeira-Ferreira A, Vieyra A
Departamento de Bioquímica, ICB, Universidade Federal do Rio de Janeiro, Brazil.
J Biol Chem. 1991 Jun 5;266(16):10249-53.
In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.
在本研究中，我们报道了钙调蛋白对肾近端小管细胞基底外侧膜上的（Ca2+ + Mg2+）-ATP酶的一种不同寻常的激活模式。去除钙调蛋白的ATP酶活性具有高Ca2+亲和力（Km = 2.2 - 3.4 microM）以及对ATP浓度的双相依赖性。该制剂对添加钙调蛋白的反应是产生一个新的具有非常高亲和力的Ca2+位点（Km小于0.05 microM）。钙调蛋白拮抗剂对ATP酶活性有多种影响。化合物48/80抑制钙调蛋白刺激的活性达70%，而氯米帕明不改变该组分。在没有钙调蛋白的情况下，48/80仍作为拮抗剂起作用，将Ca2+的Km增加到5.7 microM，并通过在低亲和力调节位点与ATP竞争来降低酶的周转率。氯米帕明不影响Ca2+亲和力，但它确实将ATP从调节位点置换出来。在固定的Ca2+（30 microM）和ATP（5 mM）浓度下，无机磷酸（Pi）可防止48/80的作用，并增强氯米帕明的抑制作用。在25 microM ATP时，Pi可防止氯米帕明的抑制作用。我们提出，ATP和Pi产生这些作用是因为药物与ATP酶的结合主要发生在E2形式上。
