Gardner P R, Fridovich I
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1987 Dec 25;262(36):17591-5.
In vitro synthesis of Escherichia coli manganese-containing superoxide dismutase, directed by the plasmid pDT1-5, has been achieved. The Mn superoxide dismutase polypeptide was identified by electrophoresis on polyacrylamide gels, immunoprecipitation, and the competitive immunoprecipitation effect of pure, active E. coli Mn superoxide dismutase. Dithiothreitol and glutathione, but not cysteine, suppressed in vitro synthesis of Mn superoxide dismutase. The parallel syntheses of beta-lactamase and of another unidentified polypeptide were not suppressed by thiols. In vitro transcription of the E. coli Mn superoxide dismutase gene was similarly suppressed by glutathione, dithiothreitol, and beta-mercaptoethanol; but not by L-cysteine or thioglycolate. Compounds, such as diamide, 1-chloro-2,4-dinitrobenzene, potassium ferricyanide, and methylene blue, which are expected to deplete intracellular glutathione, caused the induction of Mn superoxide dismutase in anaerobic E. coli.
利用质粒pDT1 - 5已实现了体外合成含锰的大肠杆菌超氧化物歧化酶。通过聚丙烯酰胺凝胶电泳、免疫沉淀以及纯的活性大肠杆菌锰超氧化物歧化酶的竞争性免疫沉淀效应鉴定了锰超氧化物歧化酶多肽。二硫苏糖醇和谷胱甘肽而非半胱氨酸抑制了锰超氧化物歧化酶的体外合成。β - 内酰胺酶和另一种未鉴定多肽的平行合成未被硫醇抑制。大肠杆菌锰超氧化物歧化酶基因的体外转录同样受到谷胱甘肽、二硫苏糖醇和β - 巯基乙醇的抑制；但不受L - 半胱氨酸或硫代乙醇酸的抑制。预期会耗尽细胞内谷胱甘肽的化合物，如二酰胺、1 - 氯 - 2,4 - 二硝基苯、铁氰化钾和亚甲蓝，会在厌氧大肠杆菌中诱导锰超氧化物歧化酶的产生。
