Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell.
The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine. An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits. With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells. KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa. The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor. We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein. With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene. The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis. Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form. Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein.
Yoshimura
A
A
Cancer Research Institute, Faculty of Medicine, Kagoshima University, Japan.
Kuwazuru
Y
Y
Sumizawa
T
T
Ikeda
S
S
Ichikawa
M
M
Usagawa
T
T
Akiyama
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007624:KB Cells; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D008562:Membrane Glycoproteins; D014415:Tunicamycin; D014268:Trifluoperazine; D014747:Vinblastine; D014700:Verapamil; D013755:Tetradecanoylphorbol Acetate