Theranostics 2018; 8(12):3275-3283. doi:10.7150/thno.25123
Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport
1. Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China
2. Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, P. R. China
3. Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P. R. China
4. State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China
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 in situ analysis of MBEs in the cellular membrane.
Methods: In this study, using membrane-bound α-glucosidase (α-Glu) as an example, we have developed a colorimetric in situ 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.
Results: The colorimetric in situ 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.
Conclusions: 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 in situ.
Keywords: membrane-bound enzyme, in situ analysis, lipid bilayer, ion transport, inhibitor evaluation
Zhang J, Li D, Yue X, Zhang M, Liu P, Li G. Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport. Theranostics 2018; 8(12):3275-3283. doi:10.7150/thno.25123. Available from http://www.thno.org/v08p3275.htm