Multidrug resistance (MDR) in human tumor cells is correlated with the presence of a 170,000 dalton membrane glycoprotein called P170 or P-glycoprotein. The complete cDNA sequence of the mdr-1 gene which encodes P170 is known. In the present study, monospecific antibodies to P170 were prepared by immunizing rabbits with 11 synthetic peptides directed against 4 major domains of P170: 1) an N-terminal intracellular domain; 2) an N-terminal extracellular domain; and (3+4) two C-terminal extracellular domains. Thus far two of these peptides have produced high titer antibodies. Antibody P7 is to domain 1 (residues # 28-35) and PO is to domain 3 (residues #741-750). Both antibodies have been affinity purified using peptide-bovine serum albumin conjugates coupled to affigel. Both antibodies specifically precipitate an (35S) methionine labeled 170 kd protein from vinblastine resistant KB cell extracts (KB-V1), which is not present in the drug-sensitive parent cell line (KB-3-1). P170 was quantitated in seven of the resistant sublines, two revertant sublines, and in two NIH-3T3 lines transfected with mdr-DNA and found to correlate with the level of drug-resistance. P170 is synthesized as a 140 kd precursor which is glycosylated slowly over the next four hrs. The oligosaccharide is asparagine-linked, endoglycosidase H resistant, and contains no sialic acid. The half- life of P170 in KB-V1 cells is 48-72 hrs, and increasing drug resistance is not due to an altered turnover rate of the protein. P170 was also detected in membrane preparations of normal human kidneys by Western blotting with the P7 antibody.
