Fruman DA, Pai SY, Klee CB, Burakoff SJ, Bierer BE
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, 02115
Methods. 1996 Apr;9(2):146-54. doi: 10.1006/meth.1996.0020.
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.
钙调磷酸酶是一种依赖钙调蛋白的磷酸酶，是免疫抑制药物环孢素A（CsA）和FK506的重要分子靶点。CsA和FK506与其亲免素配体的复合物在体外与钙调磷酸酶相互作用，导致其对多肽底物的丝氨酸/苏氨酸磷酸酶活性受到抑制。为了证明CsA和FK506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磷酸酶活性的方法。我们之前报道过，用纳摩尔浓度的CsA或FK506孵育完整细胞会导致多种细胞系中的钙调磷酸酶活性受到有效抑制。在此，我们详细讨论细胞提取物钙调磷酸酶测定法的方法和应用。
