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 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.
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 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.
Authors: I R Gibbons; M P Cosson; J A Evans; B H Gibbons; B Houck; K H Martinson; W S Sale; W J Tang Journal: Proc Natl Acad Sci U S A Date: 1978-05 Impact factor: 11.205
Authors: Sarah W Kamau; Stefanie D Krämer; Maja Günthert; Heidi Wunderli-Allenspach Journal: In Vitro Cell Dev Biol Anim Date: 2005 Jul-Aug Impact factor: 2.416