Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli.
Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli.
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从纯化的大肠杆菌硝酸还原酶中分离鉴定甲基萘醌-9。
DOI：
10.1128/jb.177.13.3728-3735.1995
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发表时间：
1995
影响因子：
3.2
通讯作者：
Dubourdieu,M
中科院分区：
文献类型：
--
作者：
Brito,F;DeMoss,JA;Dubourdieu,M
根据大肠杆菌硝酸还原酶对紫外辐射敏感的观察，其作用光谱指示萘醌（F。Brito和M. Dubourdieu，Biochem.Int.15：1079-1088，1987），我们从两种不同的纯化硝酸还原酶制剂中提取并表征了醌组分。硝酸还原酶的可溶性形式，由α和β亚基组成，纯化后释放的膜馏分通过热处理，和洗涤剂溶解的形式，含有α，β和γ（细胞色素bNR）亚基，纯化在Triton X-100的存在下。用氯仿-甲醇提取可溶性α-β-型产生了几种紫外线吸收成分，其特征是具有氧化侧链的甲基萘醌-9和甲基萘醌的进一步光破坏产物。萃取到有机相中的甲基萘醌的总量估计为0.97 mol/mol α β二聚体。通过类似的程序提取洗涤剂溶解的α β γ形式，得到两种萘醌样组分，其通过质谱表征为甲萘醌-9和去甲基甲萘醌-9的氧化形式。在这种情况下，估计总萘醌与α β二聚体的摩尔比大于6：1。当细胞色素bNR和洗涤剂从洗涤剂溶解的酶通过热处理和离子交换色谱法被消除时，只有甲基萘醌-9可以在活性α β产物的有机提取物中被识别。这些结果表明，甲基萘醌-9特异性结合的α-β-二聚体，可能是紫外线敏感的硝酸还原酶催化的电子转移途径中的组件。
On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F. Brito and M. Dubourdieu, Biochem. Int. 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase. A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100. Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone. The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer. Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9. In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1. When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product. These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.