Dataset Information

Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport.

ABSTRACT: 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.

SUBMITTER: Zhang J

PROVIDER: S-EPMC6010988 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The human voltage dependent anion channel 1 (VDAC) is a 32 kDa ?-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane ?-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

Dataset Information

Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets