ISOLATION AND IDENTIFICATION OF MENAQUINONE-9 FROM PURIFIED NITRATE REDUCTASE OF ESCHERICHIA-COLI

被引:20
作者
BRITO, F
DEMOSS, JA
DUBOURDIEU, M
机构
[1] UNIV TEXAS,SCH MED,DEPT BIOCHEM & MOLEC BIOL,HOUSTON,TX 77030
[2] UNIV LOS ANDES,FAC SCI,DEPT BIOL,MERIDA 5251,VENEZUELA
关键词
D O I
10.1128/jb.177.13.3728-3735.1995
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 (E. 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 b(NR)) subunits,,vas 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,vas estimated to be greater than 6:1. When cytochrome b(NR) 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.
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页码:3728 / 3735
页数:8
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