Plant Stress Laboratory, The Albert Katz Department of Dryland Biotechnologies, French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben-Gurion University of Negev, Sede Boqer Campus 84990, Israel.
Plant Cell Physiol. 2012 Sep;53(9):1648-58. doi: 10.1093/pcp/pcs091. Epub 2012 Jul 24.
Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.
5'-磷酸腺苷硫酸盐（APS）还原酶（APR；EC 1.8.4.9）催化 APS 的两电子还原为亚硫酸盐和 AMP，这是高等植物硫酸盐同化途径中的关键步骤。尽管这种酶很重要，但目前用于检测 APR 活性的方法依赖于放射性标记，并且只能在极少数专门配备的实验室中进行。在这里，我们提出了两种用于检测体外 APR 活性的新型动力学测定法，这些方法不需要放射性标记。在第一个测定法中，APS 用作底物，还原型谷胱甘肽（GSH）用作电子供体，而在第二个测定法中，APS 被 APS 再生系统替代，其中三磷酸腺苷硫酸化酶在反应介质中催化 APS，该系统利用硫酸盐和 ATP 作为底物。这两种动力学测定法都依赖于品红比色法检测 APR 活性的最终产物亚硫酸盐。在测定开始之前，将经过脱盐的蛋白质提取物与钨酸盐孵育，钨酸盐可抑制亚硫酸盐氧化酶活性对亚硫酸盐的氧化，从而增强实际的 APR 活性。通过测定 APR2 蛋白功能受损或过表达的拟南芥野生型和 APR 突变体的叶片提取物，验证了这两种方法的可靠性，前者缺乏 APR 活性，后者的活性比野生型高得多。该测定法还在番茄叶片上进行了测试，结果显示番茄叶片的 APR 活性高于拟南芥。所提出的 APR 测定法具有高度特异性，技术简单，并且易于在任何实验室中进行。
