REDOX STATES OF DSBA IN THE PERIPLASM OF ESCHERICHIA-COLI
REDOX STATES OF DSBA IN THE PERIPLASM OF ESCHERICHIA-COLI
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DOI：
10.1016/0014-5793(95)00354-c
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发表时间：
1995-05-01
期刊：
影响因子：
3.5
通讯作者：
ITO, K
中科院分区：
文献类型：
--
作者：
KISHIGAMI, S;AKIYAMA, Y;ITO, K
DsbA 是大肠杆菌的周质二硫键形成因子。我们研究了其活性位点半胱氨酸的体内氧化还原状态。当根据先前公布的程序从碘乙酸处理的细胞制备周质内容物时，可变但主要比例的 DsbA 处于还原形式。我们发现这是由于细胞破碎后发生的人为减少所致；即使纯化和氧化的 DsbA 在不添加任何还原剂的情况下与细胞提取物一起孵育时也会发生还原。在周质和细胞质部分中均检测到这种 DsbA 还原活性。为了避免这种伪影，我们分析了变性条件下 DsbA 的氧化还原态。现在几乎所有的 DsbA 分子都被检测为分别在 dsbB(+) 背景或 dsbB(-) 背景中氧化或还原。使用改进的方法，我们还检查了 DsbA 过量产生时的氧化还原状态，并跟踪了 DsbA 在生物合成后遵循的氧化/还原途径。这表明新合成的 DsbA 会被预先存在的 DsbA 快速氧化，而成熟（功能性）DsbA 的氧化需要 DsbB，其作用可能包括拮抗 DsbA 还原酶的作用。
DsbA is a periplasmic, disulfide bond formation factor of E. coli. We studied in vivo redox states of its active site cysteines. When periplasmic contents were prepared from iodoacetic acid-treated cells, according to the previously published procedures, variable but major proportions of DsbA were in the reduced form. We found that this was due to an artificial reduction that occurred after cell disruption; even purified and oxidized DsbA underwent reduction when incubated with cell extracts in the absence of any added reducing agent. Such DsbA-reducing activities were detected in both the periplasmic and the cytoplasmic fractions. To circumvent the artifact, we analyzed redox states of DsbA under denaturing conditions. Now virtually all the DsbA molecules were detected as oxidized or reduced in the dsbB(+) background or in the dsbB(-) background, respectively. Using the improved method, we also examined redox states of DsbA when it was overproduced, and followed the oxidation/reduction pathway that DsbA follows after biosynthesis. It is suggested that newly synthesized DsbA is rapidly oxidized by pre-existing DsbA, while oxidation of mature (functional) DsbA requires DsbB, whose roles might include that of antagonizing the actions of DsbA-reducing enzyme(s).