Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases.
Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases.
复制标题
冈田酸对蛋白磷酸酶的对硝基苯磷酸酯磷酸酶活性的抑制作用。
作者：
Akira Takai;G. Mieskes
针对碱性磷酸酶的常用底物对硝基苯基磷酸盐（pNPP）测量2A型、1型和2C型蛋白磷酸酶制剂的磷酸酶活性。在所检测的三种类型的磷酸酶中，2A型磷酸酶表现出特别高的pNPP磷酸酶活性（119 +/-8 mumol/min/mg蛋白; n = 4）。这种活性被皮科至纳摩尔浓度的冈田酸强烈抑制，冈田酸是2A型和1型蛋白磷酸酶的有效抑制剂，已被证明对碱性磷酸酶没有影响。剂量-抑制关系明显向右偏移，并随着酶浓度的增加而变得更加陡峭，正如紧密结合抑制剂的动力学理论所预测的那样。通过用冈田酸滴定估计的酶浓度与从2A型磷酸酶的蛋白质含量和分子量计算的酶浓度一致。这些结果强烈支持pNPP磷酸酶活性是2A型蛋白磷酸酶固有的，而不是由于碱性磷酸酶的污染。pNPP也被1型磷酸酶（每毫克蛋白质6.4 +/- 8 nmol/min; n = 4）和2C型磷酸酶（每毫克蛋白质1.2 +/- 3 nmol/min; n = 4）去磷酸化，但速率要低得多。1型磷酸酶制剂的pNPP磷酸酶活性显示出与其蛋白磷酸酶活性相似的对冈田酸的敏感性，而有趣的是，它对抑制剂2（1型蛋白磷酸酶的内源性抑制因子）非常耐药。2C型磷酸酶制剂的pNPP磷酸酶活性不受高达10 μ M-冈田酸的影响。
The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.