Narita Shin-ichiro, Eda Shima, Yoshihara Eisaku, Nakae Taiji
Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan.
Biochem Biophys Res Commun. 2003 Sep 5;308(4):922-6. doi: 10.1016/s0006-291x(03)01512-2.
The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).
通过免疫印迹法对铜绿假单胞菌MexAB - OprM外排泵的亚基蛋白量进行了定量。野生型菌株的单个细胞分别含有约2500、1000和1200个MexA、MexB和OprM拷贝，因此它们的化学计量比为2:1:1。mexR突变体产生的这些蛋白质水平比野生型细胞高八倍。假设MexB和OprM在泵组件中以三聚体形式存在，计算得出每个野生型细胞中MexAB - OprM的总数约为400个组件。根据完整细胞中溴化乙锭的荧光强度计算MexAB - OprM的底物外排速率，单个细胞以最大约3×10⁻¹⁹ mol s⁻¹的速率排出溴化乙锭，因此预测单个泵单元的周转率约为500 s⁻¹。
