Rossi J P, Gronda C M, Fernandez H N, Gagliardino J J
IQUIFIB - Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Biochim Biophys Acta. 1988 Aug 18;943(2):175-82. doi: 10.1016/0005-2736(88)90549-4.
Ca2+-ATPase activity was measured in rat islet homogenates, in a medium of low ionic strength containing a low concentration of Ca2+ and Mg2+ and devoid of K+. The enzyme activity was highly sensitive to inhibition by compound 48/80 (a calmodulin inhibitor), stimulated by 120 nM calmodulin and slightly affected by 10 mM NaN3. The addition of Mg2+ to the assay medium promotes the disappearance of apparent Ca2+-ATPase activity. Ouabain (0.1 mM) did not modify this ATPase activity. The enzyme showed two kinetic components for Ca2+ as well as for ATP: one with high apparent affinity and low maximum velocity and the other with low apparent affinity and high maximum velocity. Incubation of islet homogenates in this assay medium with [gamma-32P]ATP in the presence of proteolytic inhibitors, results in the appearance of a single labelled band of 130 kDa, identified by gel electrophoresis. The incorporation of 32P into this band was similar in the presence of either 2.8 or 50 microM Ca2+ and susceptible to hydroxylamine attack. The results indicate that, under the conditions described above, the Ca2+-ATPase activity evidenced in the islet homogenates had characteristics resembling those of the enzyme which catalyzes the outward Ca2+ transport. On the other hand, the method could provide a useful tool to test the effect of different agents which affect insulin secretion upon the islet plasma membrane Ca2+-ATPase activity.
在大鼠胰岛匀浆中测定Ca2+-ATP酶活性，测定介质为低离子强度，含有低浓度的Ca2+和Mg2+且不含K+。该酶活性对化合物48/80（一种钙调蛋白抑制剂）的抑制高度敏感，受120 nM钙调蛋白刺激，受10 mM叠氮化钠的影响较小。向测定介质中添加Mg2+会促使表观Ca2+-ATP酶活性消失。哇巴因（0.1 mM）不会改变这种ATP酶活性。该酶对Ca2+以及ATP均表现出两种动力学成分：一种具有高表观亲和力和低最大速度，另一种具有低表观亲和力和高最大速度。在蛋白水解抑制剂存在的情况下，将胰岛匀浆在该测定介质中与[γ-32P]ATP一起孵育，通过凝胶电泳可观察到一条130 kDa的单一标记条带。在2.8或50 μM Ca2+存在的情况下，32P掺入该条带的情况相似，且易受羟胺攻击。结果表明，在上述条件下，胰岛匀浆中显示的Ca2+-ATP酶活性具有与催化Ca2+外向转运的酶相似的特征。另一方面，该方法可为测试影响胰岛素分泌的不同试剂对胰岛质膜Ca2+-ATP酶活性的作用提供有用工具。
