Krakower G R, Kim K H
J Biol Chem. 1981 Mar 10;256(5):2408-13.
Acetyl-CoA carboxylase phosphatase has been purified from the rat epididymal fat pad. The phosphatase occurs in a complex with the carboxylase. In the purification of the phosphatase, the high molecular weight complex was initially separated by sucrose gradient centrifugation, and the phosphatase was isolated from the complex by adjusting to 80% saturation with ethanol and by chromatography on Sephadex G-75. The molecular weight of the phosphatase is 71,000 as determined by sodium dodecyl sulfate gel electrophoresis and gel chromatography on Sephacryl-200 in the presence of 6 M urea. The Km for acetyl-CoA carboxylase and glycogen phosphorylase a are 1.5 microM and 37 microM, respectively. The phosphatase has a broad substrate specificity, being active toward glycogen synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, phosphorylase a, phosphoprotamine, and p-nitrophenyl phosphate, in addition to acetyl-CoA carboxylase from fat tissue and liver. Acetyl-CoA carboxylase inhibits the dephosphorylation of phosphoprotamine, indicating that the same activity is responsible for dephosphorylating both substrates. The phosphatase requires no metal ion for activity and is not inhibited by the rat liver phosphorylase phosphatase inhibitor protein. The significance of these findings is discussed in relation to the regulation of acetyl-CoA carboxylase, and the phosphatase is compared to other phosphoprotein phosphatases.
乙酰辅酶A羧化酶磷酸酶已从大鼠附睾脂肪垫中纯化出来。该磷酸酶与羧化酶形成复合物存在。在磷酸酶的纯化过程中，高分子量复合物最初通过蔗糖梯度离心分离，然后通过用乙醇调节至80%饱和度以及在Sephadex G - 75上进行色谱分离从复合物中分离出磷酸酶。通过十二烷基硫酸钠凝胶电泳和在6 M尿素存在下在Sephacryl - 200上进行凝胶色谱测定，该磷酸酶的分子量为71,000。乙酰辅酶A羧化酶和糖原磷酸化酶a的Km分别为1.5微摩尔和37微摩尔。该磷酸酶具有广泛的底物特异性，除了对脂肪组织和肝脏中的乙酰辅酶A羧化酶有活性外，对糖原合酶、3 - 羟基 - 3 - 甲基戊二酰辅酶A还原酶、磷酸化酶a、磷酸鱼精蛋白和对硝基苯磷酸也有活性。乙酰辅酶A羧化酶抑制磷酸鱼精蛋白的去磷酸化，表明相同的活性负责两种底物的去磷酸化。该磷酸酶的活性不需要金属离子，并且不受大鼠肝脏磷酸化酶磷酸酶抑制蛋白的抑制。本文结合乙酰辅酶A羧化酶的调节对这些发现的意义进行了讨论，并将该磷酸酶与其他磷酸蛋白磷酸酶进行了比较。
