Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry
| Autor: | Michael Przybylski, Thomas Denzinger, Kurt Prof Dr Klemm, S. Haas, W Ise, Volker Gekeler, Volker Dr Figala, Wolf-Rüdiger Ulrich, C Borchers, Rainer Boer |
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| Rok vydání: | 2002 |
| Předmět: |
Dexniguldipine
Azides Dihydropyridines Antineoplastic Agents ATP-binding cassette transporter Peptide Photoaffinity Labels Tritium Mass Spectrometry Tumor Cells Cultured Humans Trypsin ATP Binding Cassette Transporter Subfamily B Member 1 Binding site Chromatography High Pressure Liquid P-glycoprotein Pharmacology chemistry.chemical_classification Binding Sites biology Photoaffinity labeling Chemistry Membrane transport protein Peptide Fragments Transport protein Biochemistry biology.protein Molecular Medicine |
| Zdroj: | Molecular Pharmacology. 61:1366-1376 |
| ISSN: | 1521-0111 0026-895X |
| DOI: | 10.1124/mol.61.6.1366 |
| Popis: | Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [(3)H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [(3)H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization. |
| Databáze: | OpenAIRE |
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