Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants.
Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants.
复制标题
DOI：
10.1016/j.ab.2017.05.026
复制
发表时间：
2017-09
影响因子：
2.9
通讯作者：
J. Smirnova;A. Fernie;C. Spahn;M. Steup
中科院分区：
文献类型：
--
作者：
J. Smirnova;A. Fernie;C. Spahn;M. Steup
麦芽糖经常作为原核和真核细胞中心碳代谢的中间产物出现。各种突变体的麦芽糖水平都很高。麦芽糖以两种异构体(α形式和β形式)形式存在，不需要酶介导的催化就可以快速相互转化。由于麦芽糖通常与其他低聚葡聚糖一起丰富，选择性定量是必不可少的。在这篇通讯中，我们提出了一种使用拟南芥4-α-葡萄糖转移酶(AtDPE2)的麦芽糖光度分析方法。在体外条件下，AtDPE2以麦芽糖为葡萄糖供体，以糖原为受体，将另一个己糖单位释放为游离糖，酶促磷酸化和氧化后光度法定量。在所使用的条件下，DPE2不会与其他二糖或寡糖发生明显的反应。与麦芽糖分析中经常使用的麦芽糖酶相比，选择性更好。两个麦芽糖异构体的还原末端相互转化处于快速平衡状态，因此，新的测定方法可以测量麦芽糖的总含量。此外，还提出了一种基于ATDPE2的连续光度分析方法，它可以定量测定β-淀粉酶的活性，并被发现优于传统的检测方法。最后，基于AtDPE2的麦芽糖测定方法被用来量化拟南芥野生型和AtDPE2缺失植株在整个光暗周期中的麦芽糖含量。这些数据与同化淀粉水平一起提供。
Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) fromArabidopsisthaliana. Underin vitroconditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify β-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels.