Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China.
Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, P. R. China.
Theranostics. 2018 May 11;8(12):3275-3283. doi: 10.7150/thno.25123. eCollection 2018.
Membrane-bound enzymes (MBEs), which make up a very high proportion of intracellular enzymes, catalyze a variety of activities that are currently analyzed by various techniques after purification. However, due to their amphipathic character, the purification of MBEs is difficult. Therefore, the most productive approach represents analysis of MBEs in the cellular membrane. In this study, using membrane-bound α-glucosidase (α-Glu) as an example, we have developed a colorimetric assay for MBEs based on the inhibitory effect of lipid bilayer on ion transport. The enzyme substrate could mediate the self-assembly of phospholipid PEG derivative around magnetic nanospheres that were modified with boronic acid. The formation of lipid bilayer could inhibit the leaking of iron ions under acidic conditions. However, the product of the catalytic reaction had no capability for self-assembly of the lipid bilayer, leading to the release of iron ions from the magnetic nanospheres under acidic pH. The colorimetric assay for MBEs could not only analyze the activity of membrane-bound α-Glu located on Caco-2 cells but could also evaluate the α-Glu inhibitors in cell medium. The simple, economic, and efficient method proposed here offers a potential application for high-throughput testing of α-Glu and its inhibitors. Our study also outlines a strategy for exploring the colorimetric method to detect the activities of MBEs .
膜结合酶（MBE）构成了细胞内酶的很大一部分，它们催化各种活性，目前通过各种技术在纯化后进行分析。然而，由于其两亲性，MBE 的纯化较为困难。因此，最有效的方法是在细胞膜中分析 MBE。在本研究中，我们以膜结合的α-葡萄糖苷酶（α-Glu）为例，开发了一种基于脂质双层对离子运输抑制作用的 MBE 比色测定法。酶底物可以介导带有硼酸盐的磁性纳米球周围的磷脂聚乙二醇衍生物的自组装。脂质双层的形成可以抑制酸性条件下铁离子的泄漏。然而，催化反应的产物没有形成脂质双层的自组装能力，导致在酸性 pH 下铁离子从磁性纳米球中释放出来。用于 MBE 的比色测定法不仅可以分析位于 Caco-2 细胞上的膜结合α-Glu 的活性，还可以评估细胞培养基中的α-Glu 抑制剂。本研究提出的这种简单、经济、高效的方法为高通量检测α-Glu 及其抑制剂提供了一种潜在的应用。我们的研究还概述了一种探索用于检测 MBE 活性的比色法的策略。
