Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells.
The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored. The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine. Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds. The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1. Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates.
Busche
R
R
Abteilung Biophysikalische Chemie, Medizinische Hochschule Hannover, Federal Republic of Germany.
Tümmler
B
B
Cano-Gauci
D F
DF
Riordan
J R
JR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000032:analysis; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003630:Daunorubicin / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004351:Drug Resistance; D005260:Female; D007700:Kinetics; D008433:Mathematics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000903:Antibiotics, Antineoplastic; D008562:Membrane Glycoproteins; C060588:iodomycin; D014747:Vinblastine; D003078:Colchicine; D003630:Daunorubicin