The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells.
CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.
Bhatia
Prateek A
PA
National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, MD 21224-6825, USA.
Moaddel
Ruin
R
Wainer
Irving W
IW
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis*; D013237:Stereoisomerism; D014700:Verapamil / Q000494:pharmacology
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D005047:Etoposide; D008024:Ligands; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D014700:Verapamil; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human; C421109:multidrug resistance-associated protein 1