Escherichia coli H+-ATPase. Glutamic acid 185 in beta subunit is essential for its structure and assembly.
Escherichia coli H+-ATPase. Glutamic acid 185 in beta subunit is essential for its structure and assembly.
复制标题
大肠杆菌 H-ATP 酶。
DOI：
10.1016/s0021-9258(18)48125-2
复制
发表时间：
1987
期刊：
影响因子：
--
通讯作者：
M. Futai
中科院分区：
文献类型：
--
作者：
T. Noumi;M. Azuma;S. Shimomura;M. Maeda;M. Futai
将大肠杆菌H+-ATPaseβ亚基UNCD基因克隆到lac启动子下游，通过寡核苷酸定向诱变(Glu-185-Gln或Lys)。将重组质粒导入缺失H+-ATPase亚单位UNC操纵子的菌株中。合成的野生型或突变型β亚基约占细胞总蛋白的10%，主要存在于细胞质中。用常规方法可以将这些亚基提纯到几乎均一。野生型和两个突变的β亚基对8-苯基-1-磺酸、金绿素和三磷酸腺苷的Kd值基本相同，尽管8-苯基-1-磺酸盐和金绿素与两个突变的β亚基结合时的荧光强度明显低于与野生型亚基结合时的荧光强度。这三个β亚基表现出基本相同的圆二色谱，表明α-螺旋含量约为16-18%。因此，突变没有引起亚基二级结构的显著变化。然而，在温度线性上升期间对theta 208的测量表明，Glu-185被Gln或Lys取代略微改变了二级结构的稳定性。使用这两个突变的β亚基只能重组微量的α-β-伽马复合体。这些结果表明，Glu-185或其附近区域可能是亚基组装所必需的。本研究建立的方法对进一步研究β亚基具有一定的参考价值。
The uncD gene for the beta subunit of Escherichia coli H+-ATPase was cloned downstream of the lac promoter and mutagenized (Glu-185—Gln or Lys) by an oligonucleotide-directed procedure. The recombinant plasmid was introduced into a strain in which the unc operon for subunits of H+-ATPase was deleted. The wild-type or mutant beta subunit synthesized amounted to about 10% total cell protein and was mainly found in the cytoplasmic fraction. These subunits could be purified to almost homogeneity by conventional procedures. The wild-type and two mutant beta subunits had essentially the same Kd values for 8-anilinonaphthalene-1-sulfonate, aurovertin, and ATP, although the fluorescence intensities of 8-anilinonaphthalene-1-sulfonate and aurovertin were significantly less when bound to the two mutant beta subunits than when bound to the wild-type subunit. The three beta subunits showed essentially the same circular dichroism spectra, indicating alpha-helical contents of about 16-18%. Thus, the mutations did not cause marked change of the secondary structure of the subunit. However, measurements of theta 208 during linear increase in temperature suggested that replacement of Glu-185 by Gln or Lys slightly changed the stability of the secondary structure. Only trace amounts of alpha beta gamma complexes could be reconstituted using the two mutant beta subunits. These results suggest that Glu-185 or the region in its vicinity may be essential for subunit assembly. The methods developed in this study should be useful for further studies on the beta subunit.