Purification and partial characterization of 200 kDa oncofetal antigen from radiation induced murine lymphocytic lymphoma.
1. A 200 kDa glycoprotein (gp200) oncofetal antigen was purified from solubilized membranes of a radiation-induced murine lymphocytic lymphoma cell line (XR11-5T), grown in syngeneic RFM mice, by successive gel chromatography of the active fraction on lentil lectin agarose, Q- and S-Sepharose and Superose-12 using an FPLC system. 2. A murine monoclonal antibody 115, produced by the syngeneic immunization of adult male C57BL/6N mice with 12-day mouse fetal cells, was used in a slot blot antibody assay to follow up the active fractions. 3. The purified glycoprotein has a pI of 5.4. 4. Treatment of radiolabeled gp200 with neuraminidase caused a slight reduction in size due to the removal of sialic acid groups and a shift in pI to 6.3. 5. Treatment of gp200 with different glycosidases shows that gp200 is susceptible to N- and O-glycanase but not to endoglycosaminidase H. 6. On extraction of gp200 with Triton X-114 it partitions exclusively into the detergent-rich fraction consistent with being an integral membrane protein.
Barsoum
A L
AL
Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile 36688.
Coggin
J H
JH
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007526:Isoelectric Point; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000737:chemistry* / Q000209:etiology; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D009381:Neoplasms, Radiation-Induced; D017830:Octoxynol; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011092:Polyethylene Glycols; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D008562:Membrane Glycoproteins; C031192:oncofetal antigens; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase