Isolated rafts from adriamycin-resistant P388 cells contain functional ATPases and provide an easy test system for P-glycoprotein-related activities.
OBJECTIVE
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 microM. 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.
Bucher
Karsten
K
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland.
Besse
Camille A
CA
Kamau
Sarah W
SW
Wunderli-Allenspach
Heidi
H
Krämer
Stefanie D
SD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007941:Leukemia P388 / Q000201:enzymology*; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D051379:Mice
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D004317:Doxorubicin; D000251:Adenosine Triphosphatases