磷蛋白磷酸酶的多种分子形式。III. 兔肝的磷酸化酶磷酸酶和磷酸组蛋白磷酸酶。

Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

Kobayaski M, Kato K, Sato S

Biochim Biophys Acta. 1975 Feb 19;377(2):343-55. doi: 10.1016/0005-2744(75)90315-0.

Abstract

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?

摘要

兔肝脏可溶性部分中催化肌肉磷酸化酶a和磷酸组蛋白（P-组蛋白）去磷酸化的磷蛋白磷酸酶（磷蛋白磷酸水解酶，EC 3.1.3.16），通过从DEAE-纤维素柱上进行NaCl梯度洗脱，被分离成三个活性部分（按洗脱顺序为部分I、II和III）。它们对两种底物具有不同的相对反应速率，并且受Mn²⁺的刺激程度也不同。部分I、II和III对磷酸化酶a的表观Km值分别为15、20和16 μM，对P-组蛋白的表观Km值分别为6.9、5.3和4.4 μM（测定混合物中含有Mn²⁺）。2. 在蔗糖密度梯度离心中，发现部分I和II含有一个主要活性峰（分别为7.0 S和7.8 S）和一个次要活性峰（4.0 S），而部分III仅含有一个峰（5.8 S）。在0.2 M巯基乙醇存在下进行冻融，使所有三个部分都解离成分子大小相似的亚基（3.4 S），同时磷酸化酶磷酸酶活性增强。Km值都基本相同（磷酸化酶a为20 μM，P-组蛋白为16 μM）。3. 用所述的任何方法都无法分离磷酸化酶磷酸酶和P-组蛋白磷酸酶活性。在一些部分中观察到两种磷蛋白底物之间的竞争。