Khandelwal R L, Enno T L
J Biol Chem. 1985 Nov 15;260(26):14335-43.
A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.
采用高速离心、酸沉淀、硫酸铵分级分离、DEAE - 纤维素柱层析、琼脂糖 - 组蛋白柱层析以及Bio - Gel A - 0.5m柱层析等方法，从兔肝脏中纯化出一种高分子量磷蛋白磷酸酶。纯化后的酶在非变性聚丙烯酰胺阴离子圆盘凝胶上呈现单一谱带，且该谱带与酶活性相关。该酶由等摩尔浓度的两个亚基组成，其分子量分别为58,000（范围58,000 - 62,000）和35,000（范围35,000 - 38,000）。另外两种多肽（分子量分别为76,000和27,000）也与我们的酶制剂紧密相关，但它们在磷酸酶活性中所起的作用（如果有）尚不清楚。该反应的最适pH值为7.5 - 8.0。磷蛋白磷酸酶对磷酸化酶a的Km值为0.10 - 0.12mg/ml。在0.2Mβ - 巯基乙醇存在下，对酶进行冻融处理会使磷酸酶活性激活（100 - 140%），同时酶会部分解离为分子量为35,000的催化亚基。二价阳离子（Mg2 +、Mn2 +和Co2 +）以及EDTA在浓度高于1mM时具有抑制作用。精胺和亚精胺在1mM浓度时也具有抑制作用。该酶受到核苷酸（ATP、ADP、AMP）、焦磷酸、磷酸以及氟化钠的抑制；每种化合物的抑制程度不同，且取决于测定中所使用的它们的浓度。在检测的各种组蛋白中，观察到III型和V型组蛋白（Sigma）对磷蛋白磷酸酶活性具有最大激活作用。对III型组蛋白的进一步研究表明，它增加了磷酸化酶a的Km值以及去磷酸化反应的Vmax。纯化的肝脏磷酸酶除了能使磷酸化酶a去磷酸化外还能催化32P标记的磷酸化酶激酶、肌球蛋白轻链、肌球蛋白、组蛋白III - S以及髓鞘碱性蛋白的去磷酸化反应。Mn2 +、KCl以及组蛋白III - S对磷酸酶活性的影响因所使用的底物而异。
