Characterization of a 95 kilodalton membrane glycoprotein associated with multi-drug resistance.
Over-expression of a 95-kDa membrane protein (P-95) has been reported previously in the multi-drug-resistant (MDR) breast cancer cell line MCF-7/AdrVp and the MDR small cell lung cancer line NCI-H1688. We have now developed anti-sera against gel-purified }-95 protein from each of these cell lines. Western blotting with each serum demonstrates a broad band at 95-kDa with detergent-solubilized membrane proteins from MCF-7/AdrVp and NCI-H1688 cells, which is barely detectable in membrane proteins from drug-sensitive parental MCF-7 cells. Each anti-serum cross-reacts with a 190-kDa membrane protein (P-190) in NCI-H1688 but not MCF-7/AdrVp cells. Immunoblotting and silver staining of NCI-H1688 membrane proteins separated by two-dimensional gel electrophoresis demonstrates that P-95 and P-190 run as broad streaks with low iso-electric points. Incubation of NCI-H1688 cells with tunicamycin or cleavage of carbohydrate residues of NCI-H1688 or MCF-7/AdrVp membrane proteins with PNGase F leads to the appearance of a sharp 35-kDa band reactive with anti-P-95 antisera. This 35-kDa protein has been isolated by two-dimensional gel electrophoresis. Neuraminidase digestion converts P-95 to a broad 65-kDa immunoreactive band, indicating the presence of terminal sialic acid residues. In conclusion, P-95 is an N-linked sialoglycoprotein with a 35-kDa polypeptide core.
Doyle
L A
LA
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Gao
Y
Y
Yang
W
W
Ross
D D
DD
D015153:Blotting, Western; D018432:Drug Resistance, Multiple; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins