Measurement of Calcineurin Phosphatase Activity in Cell Extracts
Measurement of Calcineurin Phosphatase Activity in Cell Extracts
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DOI：
10.1006/meth.1996.0020
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发表时间：
1996-04
期刊：
影响因子：
4.8
通讯作者：
Fruman;Pai;Klee;Burakoff;Bierer
中科院分区：
文献类型：
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作者：
Fruman;Pai;Klee;Burakoff;Bierer
钙调素依赖性磷酸酶钙调神经磷酸酶是免疫抑制剂环孢素A（CsA）和FK 506的重要分子靶点。CsA和FK 506与其亲免素配体的复合物在体外与钙调神经磷酸酶相互作用，导致其丝氨酸/苏氨酸磷酸酶活性对多肽底物的抑制。为了证明CsA和FK 506在体内抑制钙调磷酸酶，我们开发了一种测定粗细胞提取物中钙调磷酸酶活性的方法。我们以前曾报道，孵育的完整细胞与纳摩尔浓度的CsA或FK 506的结果在各种细胞系的钙调磷酸酶活性的有效抑制。在这里，我们详细讨论了细胞提取物钙调磷酸酶测定的方法和应用。
The calmodulin-dependent phosphatase calcineurin is an important molecular target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Complexes of CsA and FK506 with their immunophilin ligands interact with calcineurin in vitro, resulting in the inhibition of its serine/threonine phosphatase activity toward polypeptide substrates. In order to demonstrate that CsA and FK506 inhibit calcineurin in vivo, we developed an assay to measure calcineurin phosphatase activity in crude cell extracts. We have previously reported that incubation of intact cells with nanomolar concentrations of either CsA or FK506 results in potent inhibition of calcineurin activity in a variety of cell lines. Here we discuss in detail the methodology and applications of the cell extract calcineurin assay.