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Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein-Related Activities

Bucher, Karsten ; Besse, Camille A. ; Kamau, Sarah W. ; Wunderli-Allenspach, Heidi ; Krämer, Stefanie D.

In: Pharmaceutical Research, 2005, vol. 22, no. 3, p. 449-457

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    Title
    • Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein-Related Activities
    Author
    • Bucher, Karsten. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland
    • Besse, Camille A.. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland
    • Kamau, Sarah W.. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland
    • Wunderli-Allenspach, Heidi. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland
    • Krämer, Stefanie D.. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland
    Document Type
    • Postprint
    Language
    • English
    Published in
    • Pharmaceutical Research, 2005, vol. 22, no. 3, p. 449-457. Kluwer Academic Publishers-Plenum Publishers
    Other Version
    OAI-PMH Identifier
    • oai:doc.rero.ch:311172
    Summary
    No Heading: Purpose.: P-glycoprotein (P-gp), a membrane ATPase expelling many structurally unrelated compounds out of cells, is one of the major contributors to multidrug resistance. It is enriched in cold TritonX-100 insoluble membrane domains (i.e., rafts). The purpose of this work was to characterize the ATPase activities of raft preparations from P388 cells overexpressing P-gp (P388/ADR) or devoid of P-gp (P388) and to establish a P-gp-enriched screening system for P-gp-interfering compounds. Methods.: Rafts were extracted with cold TritonX-100. The ATPase activity was characterized in 96-well plates using a fluorescence assay. Results.: The ATPase activity per mg protein was about five times higher in P388/ADR rafts than in crude membranes. The anti-P-gp antibody C219 inhibited 20% of the activity in P388/ADR rafts but only about 10% of the activity in P388/ADR crude membranes and had no effect on the activity of P388 rafts. The known P-gp-activating compounds verapamil, progesterone, and valinomycin revealed the typical bell-shaped activity/concentration profiles in P388/ADR rafts, indicative for activation at low compound concentrations and inhibition at concentrations >10 to 100 μM. The inhibitory effect was also observed in P388 rafts. Conclusions.: Extracted rafts are rich in functional ATPases. Rafts from P-gp-overexpressing cells display P-gp-typical ATPase activity and provide an easy, P-gp-enriched screening system