Price D J, Tabarini D, Li H C
Eur J Biochem. 1986 Aug 1;158(3):635-45. doi: 10.1111/j.1432-1033.1986.tb09801.x.
The ATP X Mg2+-dependent phosphoprotein phosphatase has been purified from bovine heart to near-homogeneity. It is a heterodimer (75 kDa) consisting of a catalytic (C) subunit (40 kDa) and a regulatory (R) subunit (35 kDa). The R subunit, which is identical to inhibitor-2, is transiently phosphorylated during activation of the enzyme catalyzed by phosphatase-1 kinase (FA). Maximal activation requires preincubation of the phosphatase with FA and ATP X Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA X ATP X Mg activity, depending on the degree of endogenous proteolytic damage of the phosphatase during purification), but not by either FA or ATP alone. Limited trypsinization results in a rapid and total degradation of the R subunit and partial degradation of the 40-kDa C subunit to active proteins of 35-38 kDa. The resulting 'nicked' C subunit of 35-38 kDa is no longer dependent on FA for activation and can be fully activated by Mg2+ (or Mn2+) alone. Endogenous proteolytic damage of the R subunit also results in an increase of activity that can be expressed by M2+ alone with a concomitant decrease of the FA-dependent activation. Although Mn2+ is slightly more effective than Mg2+ in expressing the holoenzyme basal activity, the activation by Mn2+ is only about 60% of that of Mg2+ when FA and ATP are also present. In the activation by adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), Co2+ is the most effective cofactor. The activation by ATP[gamma S] X Co2+ is more than 50% of that by ATP X Mg2+. The present studies indicate that Mg2+ is the natural divalent cation for the FA-catalyzed activation in which Mg2+ plays two distinctly different roles: it forms Mg2+ X ATP which serves as a substrate for the kinase; it acts as an essential cofactor for the catalytic function of the phosphatase. The discrepancies between the results obtained by this and other laboratories with respect to the effectiveness of Mg2+ and ATP[gamma S] in the activation of the phosphatase are discussed.
ATP 与 Mg2+ 依赖的磷蛋白磷酸酶已从牛心脏中纯化至接近均一。它是一种异源二聚体（75 kDa），由催化（C）亚基（40 kDa）和调节（R）亚基（35 kDa）组成。R 亚基与抑制剂 -2 相同，在磷酸酶 -1 激酶（FA）催化的酶激活过程中会短暂磷酸化。最大激活需要磷酸酶与 FA 和 ATP 与 Mg2+ 预孵育。然而，单独的 Mg2+ 即可表达相对较低但明确可证明的基础活性（占 FA 与 ATP 与 Mg 活性的 3% 至 10%，具体取决于纯化过程中磷酸酶内源性蛋白水解损伤的程度），但单独的 FA 或 ATP 则不能。有限的胰蛋白酶消化导致 R 亚基快速完全降解以及 40 kDa 的 C 亚基部分降解为 35 - 38 kDa 的活性蛋白。产生的 35 - 38 kDa 的“切口”C 亚基不再依赖 FA 激活，仅 Mg2+（或 Mn2+）即可将其完全激活。R 亚基的内源性蛋白水解损伤也导致活性增加，这种活性可单独由 M2+ 表达，同时 FA 依赖激活降低。尽管 Mn2+ 在表达全酶基础活性方面比 Mg2+ 稍有效，但当同时存在 FA 和 ATP 时，Mn2+ 的激活仅约为 Mg2+ 的 60%。在由腺苷 5'-[γ - 硫代]三磷酸（ATP[γS]）激活时，Co2+ 是最有效的辅因子。ATP[γS] 与 Co2+ 的激活超过 ATP 与 Mg2+ 激活的 50%。本研究表明，Mg2+ 是 FA 催化激活的天然二价阳离子，其中 Mg2+ 发挥两种截然不同的作用：它形成 Mg2+ 与 ATP，作为激酶的底物；它作为磷酸酶催化功能的必需辅因子。讨论了本实验室与其他实验室在 Mg2+ 和 ATP[γS] 激活磷酸酶有效性方面所得结果的差异。
