PROPERTIES AND SEQUENCE OF THE NHAA NA+/H+ ANTIPORTER OF VIBRIO-PARAHAEMOLYTICUS
PROPERTIES AND SEQUENCE OF THE NHAA NA+/H+ ANTIPORTER OF VIBRIO-PARAHAEMOLYTICUS
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DOI：
10.1093/oxfordjournals.jbchem.a124624
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发表时间：
1994-11-01
影响因子：
2.7
通讯作者：
TSUCHIYA, T
中科院分区：
文献类型：
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作者：
KURODA, T;SHIMAMOTO, T;TSUCHIYA, T
从微嗜盐海洋细菌副溶血性弧菌的染色体DNA中克隆出Na+/H+反转运蛋白基因。宿主是大肠杆菌突变体，缺乏两种主要的Na+/H+反转运蛋白NhaA和NhaB。未转化的突变细胞在0.6 M NaCl或0.1 M LiCl存在下无法生长，但Na+和Li+对携带反转运基因的质粒转化的细胞无毒。从原始大肠杆菌突变体制备的膜囊泡没有显示出任何可检测的Na+/H+（和Li+/H+）反端口活性。然而，我们观察到转化细胞制备的膜囊具有较高的Na+/H+（和Li+/H+）抗运活性。当实验培养基的pH从7.0增加到8.5时，活性显著增加。这种性质与大肠杆菌的NhaA Na+/H+反转运蛋白非常相似。与副溶血性弧菌囊泡相比，转化细胞制备的膜囊泡中Li+和Na+的K-m值急剧下降。克隆基因的核苷酸序列与大肠杆菌的NhaA Na+/H+反转运蛋白具有高度的同源性（同源性59%，相似性87%）。因此，我们得出结论，我们克隆和测序的基因是副溶血性弧菌的nhaA。我们还发现NhaA蛋白的几个区域与其他一些生物体的转运蛋白序列相似。这些区域似乎对Na+识别、转运或酰胺结合很重要。
A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of the slightly halophilic marine bacterium Vibrio parahaemolyticus. The host was an Escherichia coli mutant that lacked both of the two major Na+/H+ antiporters, NhaA and NhaB. Untransformed mutant cells were unable to grow in the presence of 0.6 M NaCl or 0.1 M LiCl, but Na+ and Li+ were non-toxic to cells transformed with a plasmid carrying the antiporter gene. Membrane vesicles prepared from the original E. coli mutant did not show any detectable Na+/H+ (and Li+/H+) antiport activity. However, we observed high Na+/H+ (and Li+/H+) antiport activity in membrane vesicles prepared from the transformed cells. The activity increased greatly when the pH of the assay medium was increased from 7.0 and 8.5. This property is very similar to that of the NhaA Na+/H+ antiporter of E. coli. Drastic decreases in K-m values for Li+ and Na+ were observed with membrane vesicles prepared from the transformed cells compared with those observed with V. parahaemolyticus vesicles. The amino acid sequence deduced from the nucleotide sequence of the cloned gene showed high homology (59% identity and 87% similarity) with the NhaA Na+/H+ antiporter of E. coli. Thus, we conclude that the gene we cloned and sequenced is the nhaA of V. parahaemolyticus. We also found that several regions of the NhaA protein showed sequence similarity with transport proteins from some other organisms. Such regions seem to be important for Na+ recognition, transport or amiloride binding.