Disulfide bonding of a human prostate tumor-associated membrane antigen recognized by monoclonal antibody D83.21.
Previous reports from our laboratory have described the binding specificity of a monoclonal antibody, D83.21, prepared by fusing P3X63/Ag8 mouse myeloma cells with mouse lymphocytes immunized against the DU145 human prostate adenocarcinoma cell line. The D83.21 monoclonal antibody displayed preferential binding to human prostate and bladder carcinoma cell lines and tissues. This antibody was not reactive with a variety of other normal and malignant human cell lines or tissues. Immunofluorescence analysis indicated that the D83.21 antigen was located on the plasma membrane. Biochemical characterization of the target antigen was performed by subjecting detergent-soluble extracts of [3H]glucosamine-labeled cells to D83.21 monoclonal antibody affinity chromatography. The radioactive material that was specifically bound and eluted from the affinity column was analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and fluorography. In the absence of 2-mercaptoethanol, the antigen displayed two prominent bands with molecular weights of 180 and 110 kilodaltons. In the reduced form, the antigen is composed of an Mr 60,000 heavy chain and an Mr 28,000 light chain. The antigen was further resolved using two-dimensional (intact/reduced) sodium dodecyl sulfate: polyacrylamide gel electrophoresis. These analyses indicated that the Mr 180,000 chain was composed entirely of Mr 60,000 subunits, whereas the Mr 110,000 band consisted only of Mr 28,000 subunits. The antigen recognized by the D83.21 monoclonal antibody is therefore a membrane glycoprotein with a subunit structure cross-linked together through disulfide bonds.
Starling
J J
JJ
Wright
G L
GL
D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008970:Molecular Weight; D017430:Prostate-Specific Antigen; D011471:Prostatic Neoplasms / Q000276:immunology*
D000951:Antigens, Neoplasm; D004220:Disulfides; D017430:Prostate-Specific Antigen