Jeyaseelan K, Guest J R, Visser J
J Gen Microbiol. 1980 Oct;120(2):393-402. doi: 10.1099/00221287-120-2-393.
The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (El; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77800) and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 micromol substrate transformed (mg protein)-1 min-1 at the optimum pH (7.8) and 25 degrees C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated ace A mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, and ace B mutant, lacked the activity of the acetyltransferase (E2 component).
通过在乙醇-琼脂糖2B上进行亲和层析，随后进行蔗糖密度梯度离心，对铜绿假单胞菌PAO的丙酮酸脱氢酶复合物进行了纯化。基于酶活性，总体纯化倍数为130倍。当通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时，纯化的复合物含有三个主要和一个次要的多肽组分。通过热处理、有限蛋白酶解和肽图谱分析，这些组分被鉴定为丙酮酸脱氢酶（E1；分子量92500）、乙酰转移酶（E2；主要组分，分子量76000，次要组分，分子量77800）和硫辛酰胺脱氢酶（E3；分子量58000）。纯化的复合物沉降系数为48S，在最佳pH（7.8）和25℃下，复合物总体反应的比活性为6.5微摩尔底物转化（毫克蛋白）-1分钟-1。在对相应的无细胞提取物进行部分纯化后，鉴定了四个缺乏总体丙酮酸脱氢酶复合物活性的ace突变体中的损伤。三个菌株，指定为ace A突变体，缺乏丙酮酸脱氢酶活性（E1组分），一个菌株，即ace B突变体，缺乏乙酰转移酶（E2组分）的活性。
