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Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as
1Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany. W.Hagmann@DKFZ-Heidelberg.de
Abstract:
The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.
Insights
Researchers purified the multidrug resistance-associated protein 2 (MRP2) for functional studies. The purified MRP2 protein exhibited ATPase activity and transported glutathione conjugates, providing insights into its role in drug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The multidrug resistance-associated protein 2 (MRP2), also known as ABCC2, is an ATP-dependent efflux pump crucial for cellular drug resistance.
- Understanding MRP2's function requires purified, active protein for detailed biochemical and biophysical analysis.
Purpose of the Study:
- To develop a method for the purification and functional characterization of MRP2 (ABCC2).
- To investigate the ATPase activity and substrate transport capabilities of purified MRP2.
Main Methods:
- Stable expression of C-terminally (His)6-tagged MRP2 in HEK293 cells.
- Solubilization, purification using affinity chromatography, and MS verification of MRP2.
- Reconstitution of purified MRP2 into proteoliposomes for functional assays.
Main Results:
- Successfully expressed and purified functional MRP2-(His)6.
- Purified MRP2 exhibited ATP-dependent ATPase activity, stimulated by glutathione conjugates.
- Reconstituted MRP2 transported leukotriene C4, confirming its conjugate export function.
Conclusions:
- The study provides a method for obtaining pure, functional MRP2.
- Purified MRP2's ATPase and transport activities offer a platform for future studies on its role in multidrug resistance and cellular transport mechanisms.