大肠杆菌提取物中腺苷酸环化酶调节特性的重建。
Reconstitution of regulatory properties of adenylate cyclase in Escherichia coli extracts.
作者信息
Reddy P, Meadow N, Roseman S, Peterkofsky A
出版信息
Proc Natl Acad Sci U S A. 1985 Dec;82(24):8300-4. doi: 10.1073/pnas.82.24.8300.
The inhibition of adenylate cyclase activity of Escherichia coli by methyl alpha-glucoside has been demonstrated in intact or in permeable cells but not in cell-free extracts. In intact or permeable cells, this inhibition is demonstrable only in strains expressing the genes for proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS); in permeable cells, the inhibition also requires potassium phosphate. Using homogeneous proteins of the PTS, we have reconstituted in cell-free extracts many of the features of the regulated form of adenylate cyclase: (i) In the absence of K2HPO4, permeable cells have lower adenylate cyclase activity than extracts; addition of homogeneous PTS proteins to the extracts brings adenylate cyclase activity close to the level observed in permeable cells. (ii) The low activity observed in permeable cells is stimulated by potassium phosphate; this stimulation is also observed in extracts supplemented with PTS proteins and phosphoenolpyruvate. (iii) In permeable cells, potassium phosphate-stimulated adenylate cyclase activity is inhibited by methyl alpha-glucoside or pyruvate; extracts behaved similarly when supplemented with PTS proteins, K2HPO4, and phosphoenolpyruvate. Thus, the regulated form of adenylate cyclase has been reconstituted in cell-free extracts by addition of homogeneous PTS proteins.
α-甲基葡萄糖苷对大肠杆菌腺苷酸环化酶活性的抑制作用已在完整细胞或通透细胞中得到证实，但在无细胞提取物中未得到证实。在完整细胞或通透细胞中，这种抑制作用仅在表达磷酸烯醇丙酮酸：葡萄糖磷酸转移酶系统（PTS）蛋白基因的菌株中可检测到；在通透细胞中，这种抑制作用还需要磷酸钾。利用PTS的纯蛋白，我们在无细胞提取物中重建了腺苷酸环化酶调节形式的许多特性：（i）在没有K2HPO4的情况下，通透细胞的腺苷酸环化酶活性低于提取物；向提取物中添加纯PTS蛋白可使腺苷酸环化酶活性接近通透细胞中观察到的水平。（ii）通透细胞中观察到的低活性受到磷酸钾的刺激；在补充了PTS蛋白和磷酸烯醇丙酮酸的提取物中也观察到这种刺激。（iii）在通透细胞中，磷酸钾刺激的腺苷酸环化酶活性受到α-甲基葡萄糖苷或丙酮酸的抑制；当补充PTS蛋白、K2HPO4和磷酸烯醇丙酮酸时，提取物的表现类似。因此，通过添加纯PTS蛋白，在无细胞提取物中重建了腺苷酸环化酶的调节形式。
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