Wingender-Drissen R, Becker J U
Biochim Biophys Acta. 1983 Mar 30;743(3):343-50. doi: 10.1016/0167-4838(83)90392-8.
Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).
通过对酵母提取物进行二乙氨基乙基纤维素（DEAE -纤维素）色谱分析，分离出了作用于肌肉磷酸化酶（1,4-α-D-葡聚糖：正磷酸α-D-葡糖基转移酶，EC 2.4.1.1）的蛋白磷酸酶（磷酸蛋白磷酸水解酶，EC 3.1.3.16）活性的三个峰值（组分a、b和c）。与组分a和b不同，只有组分c能够从32P标记的失活酵母磷酸化酶中释放磷酸盐。组分c对两种底物的活性完全依赖于二价金属离子（Mg2+、Mn2+）的存在，并被Mg·ATP激活。在巯基乙醇存在下冷冻后，组分a和b也能够使酵母磷酸化酶去磷酸化。兔肌肉磷酸蛋白磷酸酶抑制剂1和2表明，作用于肌肉磷酸化酶的酵母磷酸酶被抑制剂2抑制，但不被抑制剂1抑制。组分c对酵母磷酸化酶的作用不受任何一种抑制剂的抑制。通过离子交换色谱、酪蛋白-琼脂糖凝胶色谱和葡聚糖G - 200凝胶过滤，将天然酵母磷酸化酶磷酸酶（EC 3.1.3.17）纯化了8000倍。纯化后的酶无法使兔肌肉磷酸化酶a去磷酸化，但作用于磷酸酪蛋白（Km为3.3 mg/ml）。估计分子量为78000，最适pH为6.5 - 7.5。该酶的活性依赖于二价金属离子（Mg2+、Mn2+），并被氟化物（Ki为20 mM）和琥珀酸盐（Ki为10 mM）抑制。
