Miller E, Fredholm B, Miller R E, Steinberg D, Mayer S E
Biochemistry. 1975 Jun 3;14(11):2470-80. doi: 10.1021/bi00682a029.
Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.
采用乙醇沉淀、DEAE - 纤维素吸附、AMP - 琼脂糖亲和层析和琼脂糖凝胶过滤法，将猪脂肪组织中的糖原磷酸化酶纯化了近700倍。纯化后的酶在聚丙烯酰胺凝胶电泳中呈现为一个主要成分和几个次要成分。活性与主要成分以及至少一个次要成分相关。在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳估算，解离、还原和烷基化后的酶分子量为90,000。在AMP存在下，纯化酶的稳定性显著提高。粗匀浆中该酶的等电点pH为6.3。通过蔗糖密度梯度沉降法测定了纯化酶（7.9 S）和粗匀浆中酶（7.3 S）的沉降系数。酶活性的最适pH在6.5至7.1之间。糖原和无机磷酸的表观Km值分别为0.9 mg/ml和6.6 mM。AMP的Ka为0.21 mM。K2SO4、KF、KCl和MgCl2可提高酶活性，而NaCl、Na2SO4、D - 葡萄糖和ATP则降低酶活性。葡萄糖的抑制作用与激活剂AMP无竞争性；ATP的抑制作用与AMP部分竞争性。纯化后的酶与骨骼肌磷酸化酶激酶一起温育可被激活。粗匀浆中的酶通过添加MgCl2和ATP被激活；添加蛋白激酶抑制剂并不能阻断激活，这表明猪脂肪组织匀浆中的磷酸化酶激酶在很大程度上以激活形式存在。使用通过乙醇沉淀、DEAE - 纤维素层析和凝胶过滤从猪脂肪组织中纯化约200倍的酶，研究了磷酸化酶磷酸酶对磷酸化酶a的失活作用。脂肪组织磷酸酶对骨骼肌磷酸化酶a的Km为6 μM。用32 - P标记的纯化猪脂肪组织磷酸化酶被脂肪组织磷酸酶失活并去磷酸化。AMP抑制骨骼肌和脂肪组织底物的去磷酸化作用，葡萄糖可逆转这种抑制。几条证据表明，AMP的抑制作用是由于对底物而非对酶的作用。我们之前报道过，大鼠脂肪细胞中磷酸化酶激活系统在一些重要特征上与骨骼肌中的不同。然而，猪脂肪磷酸化酶和磷酸化酶磷酸酶的主要特性与骨骼肌中描述的酶非常相似。
