Zevgolis V G, Sotiroudis T G, Evangelopoulos A E
Institute of Biological Research, National Hellenic Research Foundation, Athens, Greece.
Biochim Biophys Acta. 1991 Jan 31;1091(2):222-30. doi: 10.1016/0167-4889(91)90065-6.
Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.
通过涉及DEAE - 纤维素色谱、硫酸铵分级分离和甘油密度超速离心的方法，从牛胃平滑肌中纯化了磷酸化酶激酶（110倍）。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）上，最终的酶制剂显示出一条43 kDa的单一蛋白带。氨基酸分析和胰蛋白酶解十肽片段测序表明，纯化的蛋白质与牛主动脉肌动蛋白有密切的相似性，尽管在几个方面与肌动蛋白有很大差异。为了将磷酸化酶激酶活性与43 kDa的蛋白质分离，我们使用了多种色谱方法，但在所有情况下，催化活性（洗脱时）都伴随着43 kDa的蛋白带。牛胃磷酸化酶激酶的表观分子量为950 kDa，它对磷酸化酶b的Vmax值较低（85 nmol·min⁻¹·mg⁻¹），pH 6.8/8.2时的活性比为0.23，它绝对需要Ca²⁺，并且被Ca²⁺/钙调蛋白激活1.8倍。此外，该蛋白激酶活性既不受抗兔骨骼肌磷酸化酶激酶抗体的抑制，也不受蛋白质磷酸化的激活。这些结果表明，牛胃磷酸化酶激酶与肌动蛋白样分子的聚集体紧密结合。
