In vitro characterization of a plastid terminal oxidase (PTOX)
In vitro characterization of a plastid terminal oxidase (PTOX)
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DOI：
10.1046/j.1432-1033.2003.03766.x
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发表时间：
2003-09-01
期刊：
影响因子：
--
通讯作者：
Kuntz, M
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文献类型：
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作者：
Josse, EM;Alcaraz, JP;Kuntz, M
在大肠杆菌细胞中表达由拟南芥IMMUTANS基因编码的质体末端氧化酶（PTOX），并使用NADH作为电子供体在分离的细菌膜中监测其醌/氧氧化还原酶活性。观察到对质体醌的特异性。泛醌、杜醌、叶绿醌和苯醌均不能代替质体醌。然而，杜醌醇（完全化学还原）是一种可接受的底物。铁也是必需的，不能被Cu 2+、Zn 2+或Mn 2+取代。这种质体醌醇氧化酶的活性在15- 40 ℃范围内与温度无关，但随pH值（从5.5到9.0）而增加。与高等植物线粒体交替氧化酶不同，PTOX显示序列相似性（但也有差异，特别是在推定的醌结合位点和半胱氨酸保守性方面），PTOX活性似乎不受丙酮酸或任何其他测试糖的调节，也不受AMP的调节。然而，其活性随着盐（NaCl或KCl）浓度的增加而降低。测试了各种醌类似物对PTOX的抑制活性。发现连苯三酚类似物是抑制剂，尤其是没食子酸辛酯（I-50=0.4 μ M），其似乎比没食子酸丙酯或没食子酸有效得多。因此，没食子酸辛酯是一种有用的抑制剂，用于未来的体内或细胞器研究，旨在研究PTOX在氯呼吸中的作用，并作为类胡萝卜素生物合成的辅因子。
The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu2+, Zn2+ or Mn2+. This plastoquinol oxidase activity is independent of temperature over the 15-40degreesC range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I-50=0.4 muM) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.