Kasai K, Field J B
Metabolism. 1983 Mar;32(3):296-307. doi: 10.1016/0026-0495(83)90196-8.
Phosphoprotein phosphatases (phosphoprotein phosphohydrolase, EC 3.1.3.16) were partially purified from bovine thyroid with phosphorylated mixed histones, H1 histone and casein as substrates. Utilizing DEAE-cellulose chromatography, (NH4)2SO4 precipitation, gel filtration before and after freeze-thawing in 0.2 M 2-mercaptoethanol and histone-Sepharose chromatography, four fractions of enzyme activity were obtained and were designated as phosphatases I, IIA, IIB, and III. Phosphatases I had an apparent molecular weight of 155,000 and was dependent on Mn2+ for maximal activity. The enzyme had the greatest activity with histone H1 and was greatly stimulated by NaCl with phosphohistones as substrate. Phosphatases IIA and IIB had a molecular weight of about 70,000, were stimulated over 5-fold by Mn2+ and had much higher activities with phosphohistones than with casein in the presence of the cation. Phosphatase III, a possible catalytic subunit of larger molecular weight forms, had an apparent molecular weight of 30,000, was generally independent of Mn2+ and had high activities using all three substrates. Phosphatases I, IIA, and III were inhibited in a dose-dependent manner by sodium pyrophosphate (PPi), ATP, potassium phosphate (Pi) and sodium fluoride (NaF) when they were added directly to the reaction mixture with phosphorylated mixed histones as substrate. PPi was the most potent inhibitor and phosphatase III was the most sensitive to inhibition. PPi, ATP and NaF probably inactivated phosphatase III activity by removing an essential metal ion. After extensive dialysis to remove these inhibitors, the inactivated enzyme could be fully activated by Mn2+, but not by Mg2+, Ba2+, Cu2+, Cd2+, Ca2+, Zn2+ and Fe2+. Whereas the enzyme pretreated with Pi retained about 80% activity after dialysis, its activity was not further stimulated by Mn2+. The inactivated (demetallized) enzyme was less reactivated by Mn2+ in the presence of mM concentration of Pi. Moreover, the Mn2+-reactivated enzyme was again inactivated by Pi, NaF and ATP. Among them Pi was the most potent inactivator. These results suggest that Pi may have another inhibitory effect on metal ion binding besides on substrate binding and also that phosphatase III might be a metalloenzyme. In bovine thyroid, there are at least two major phosphoprotein phosphatases which may have different properties. Metal ion stimulation of phosphatase I and IIA activities may be through an interaction with the substrate or with a metal ion binding site on the regulatory subunit. The lowest molecular weight enzyme (phosphatase III) probably does not exist naturally in the cell.
以磷酸化混合组蛋白、H1组蛋白和酪蛋白为底物，从牛甲状腺中部分纯化了磷蛋白磷酸酶（磷蛋白磷酸水解酶，EC 3.1.3.16）。利用DEAE - 纤维素色谱法、硫酸铵沉淀法、在0.2 M 2 - 巯基乙醇中冻融前后的凝胶过滤法以及组蛋白 - 琼脂糖色谱法，获得了四个酶活性组分，分别命名为磷酸酶I、IIA、IIB和III。磷酸酶I的表观分子量为155,000，其最大活性依赖于Mn2 +。该酶对组蛋白H1的活性最高，以磷酸化组蛋白为底物时，NaCl可极大地刺激其活性。磷酸酶IIA和IIB的分子量约为70,000，在Mn2 +的刺激下活性提高超过5倍，在阳离子存在的情况下，它们对磷酸化组蛋白的活性比对酪蛋白的活性高得多。磷酸酶III可能是较大分子量形式的催化亚基，其表观分子量为30,000，通常不依赖于Mn2 +，并且对所有三种底物都有高活性。当将焦磷酸钠（PPi）、ATP、磷酸钾（Pi）和氟化钠（NaF）直接添加到以磷酸化混合组蛋白为底物的反应混合物中时，磷酸酶I、IIA和III以剂量依赖性方式受到抑制。PPi是最有效的抑制剂，磷酸酶III对抑制最敏感。PPi、ATP和NaF可能通过去除必需的金属离子而使磷酸酶III失活。经过广泛透析以去除这些抑制剂后，失活的酶可以被Mn2 +完全激活，但不能被Mg2 +、Ba2 +、Cu2 +、Cd2 +、Ca2 +、Zn2 +和Fe2 +激活。而用Pi预处理的酶在透析后保留了约80%的活性，其活性不再被Mn2 +进一步刺激。在毫摩尔浓度的Pi存在下，失活（脱金属）的酶被Mn2 +再激活的程度较低。此外，Mn2 +再激活的酶又被Pi、NaF和ATP失活。其中Pi是最有效的失活剂。这些结果表明，Pi除了对底物结合有抑制作用外，可能对金属离子结合还有另一种抑制作用，并且磷酸酶III可能是一种金属酶。在牛甲状腺中，至少有两种主要的磷蛋白磷酸酶，它们可能具有不同的性质。金属离子对磷酸酶I和IIA活性的刺激可能是通过与底物或调节亚基上的金属离子结合位点相互作用实现的。分子量最低的酶（磷酸酶III）可能在细胞中不是天然存在的。
