Characteristics of calmodulin binding to purified human lymphocyte plasma membranes.
Characteristics of calmodulin binding to purified human lymphocyte plasma membranes.
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DOI：
10.4049/jimmunol.139.1.42
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发表时间：
1987-07
影响因子：
4.4
通讯作者：
T. P. Lee;J. Venuti;I. Macara;R. Kawauchi;P. Davis;B. Mookerjee
中科院分区：
文献类型：
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作者：
T. P. Lee;J. Venuti;I. Macara;R. Kawauchi;P. Davis;B. Mookerjee
我们探讨了钙调素的作用，在质膜相关的现象，在淋巴细胞活化的测量[125 I]钙调素结合高度纯化的人外周血淋巴细胞的质膜。钙依赖性钙调素结合淋巴细胞膜被发现在37 ° C下孵育5分钟内达到平衡，并且是可饱和的和特异性的。确定了一类高亲和力结合位点，解离常数（Kd）为1至3 × 10（-8）M，总结合容量（Bt）为1至2 pmol/mg膜蛋白。半数最大结合所需的游离钙浓度为100至300 nM。这是惊人的相似的细胞质游离钙活性[Ca 2 +]i测量的Quin-2荧光技术，特别是在刺激后与植物促分裂剂。钙调素结合可被三氟拉嗪（TFP）、W-7和氯丙嗪（所有这些都是钙调素拮抗剂）抑制。发现引起对植物分裂原的淋巴细胞增殖反应的50%抑制的TFP浓度与引起钙调蛋白与淋巴细胞质膜结合的50%抑制的TFP浓度相同。SDS-聚丙烯酰胺凝胶电泳，然后凝胶覆盖和放射自显影与碘化钙调蛋白显示五个钙依赖性，TFP-reproditable，钙调蛋白结合多肽。
We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides.