Iwasaki H, Shiba T, Makino K, Nakata A, Shinagawa H
Department of Experimental Chemotherapy, Osaka University, Japan.
J Bacteriol. 1989 Oct;171(10):5276-80. doi: 10.1128/jb.171.10.5276-5280.1989.
The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.
大肠杆菌的ruvA和ruvB基因构成一个属于SOS调控子的操纵子。遗传学证据表明，ruv操纵子的产物参与DNA修复和重组。为了开始对这些蛋白质进行生化特性分析，我们开发了一种质粒系统，该系统可使RuvB蛋白的产量增加到总细胞蛋白的20%。从过量表达系统开始，我们纯化了RuvB蛋白。纯化后的RuvB蛋白在凝胶过滤色谱中表现为单体，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中的表观相对分子质量为38千道尔顿，这与从DNA序列预测的值一致。对纯化蛋白的氨基末端区域的氨基酸序列进行了分析，该序列与从DNA序列推导的序列一致。由于RuvB蛋白的推导序列包含ATP结合蛋白的共有序列，我们检测了纯化的RuvB蛋白的ATP结合和ATP酶活性。RuvB蛋白对ADP的亲和力比对ATP的亲和力更强，且ATP酶活性较弱。结果表明，RuvB蛋白的弱ATP酶活性至少部分是由于ADP的终产物抑制作用。
