Manalan A S, Werth D K
Circ Res. 1987 Apr;60(4):602-11. doi: 10.1161/01.res.60.4.602.
A calmodulin-stimulated protein phosphatase has been purified from bovine myocardium. The purification procedure involves sequential DEAE-Sephacel ion exchange chromatography, calmodulin-Sepharose affinity chromatography, and high performance liquid chromatography using a Spherogel TSK DEAE 5PW column. By SDS polyacrylamide gel electrophoresis, the purified cardiac phosphatase consists of two subunits of Mr 61,000 and 19,000, similar to the brain enzyme, calcineurin. Protein phosphatase activity of the cardiac enzyme is stimulated by Ca2+-calmodulin and inhibited by the calmodulin antagonist drug, calmidazolium. Effects of a series of divalent cations on catalytic activity of the cardiac calmodulin-stimulated protein phosphatase are similar to those observed with calcineurin, when the two enzymes are assayed under identical conditions. Highly enriched preparations of bovine cardiac sarcolemma contain substrates of cAMP-dependent protein kinase of Mr 166 K, 133 K, 108 K, 79 K, 39 K, and 14 K, which are specifically dephosphorylated by the calmodulin-stimulated phosphatase with pseudofirst-order rate constants of 0.23, 0.46, 0.69, 0.35, 0.69, and 0.115 min-1, respectively. These substrates are not present in purified preparations of cardiac sarcoplasmic reticulum. These results support a role of the calmodulin-stimulated phosphatase in the Ca2+-regulation of specific sarcolemmal processes by protein dephosphorylation.
一种钙调蛋白刺激的蛋白磷酸酶已从牛心肌中纯化出来。纯化过程包括依次进行DEAE-琼脂糖凝胶离子交换色谱、钙调蛋白-琼脂糖亲和色谱以及使用Spherogel TSK DEAE 5PW柱的高效液相色谱。通过SDS聚丙烯酰胺凝胶电泳，纯化的心脏磷酸酶由分子量为61,000和19,000的两个亚基组成，与脑酶钙调神经磷酸酶相似。心脏酶的蛋白磷酸酶活性受到Ca2+-钙调蛋白的刺激，并被钙调蛋白拮抗剂药物氯咪达唑抑制。当在相同条件下测定这两种酶时，一系列二价阳离子对心脏钙调蛋白刺激的蛋白磷酸酶催化活性的影响与钙调神经磷酸酶观察到的相似。高度富集的牛心脏肌膜制剂含有分子量为166K、133K、108K、79K、39K和14K的cAMP依赖性蛋白激酶的底物，这些底物被钙调蛋白刺激的磷酸酶特异性去磷酸化，假一级速率常数分别为0.23、0.46、0.69、0.35、0.69和0.115 min-1。这些底物不存在于纯化的心脏肌浆网制剂中。这些结果支持钙调蛋白刺激的磷酸酶在通过蛋白去磷酸化对特定肌膜过程进行Ca2+调节中的作用。
