Murine cell surface glycoproteins. Identification, purification, and characterization of a major glycosylated component of 110,000 daltons by use of a monoclonal antibody.
A 110,000-dalton plasma membrane glycoprotein of mouse cells has been identified, purified, and characterized by use of a xenogeneic monoclonal antibody. The glycoprotein was a major component of the NIH/3T3 cell surface. It contained 15% of [3H]glucosamine incorporated into cell proteins and was composed of at least 16 isomorphic variants with apparent molecular weights from 100,000 to 120,000 and isoelectric points between 6.5 and 8.1. The glycoprotein constituted 0.1% of total cell protein, as measured by the yield of purified protein, and there were over 10(6) antibody-binding sites/cell. Immunoprecipitation from pulse-chase labeled cells showed that the 110,000-dalton glycoprotein was initially synthesized as a 92,000-dalton microsomal polypeptide which gradually was converted to the mature surface form. The turnover time of the surface form was at least 20 h. The antigenic determinant recognized by the monoclonal antibody was species-specific and nonpolymorphic. It was present in high concentration on most dividing murine cells in culture, whereas the concentration in normal mouse tissues differed widely. Among lymphoid tissues, antigen concentration was enriched in bone marrow as compared to spleen and thymus. Among nonlymphoid tissues, antigen concentration was 10-fold greater in kidney than in brain, liver, and skeletal muscle. The glycoprotein was purified without loss of antigenic activity by antibody affinity chromatography. The single step procedure yielded 3 mg of pure glycoprotein from 3 g of crude cell extract. The purification and characterization of this major membrane protein provide a basis for further study of its cell surface structure and function.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000302:isolation & purification; D002462:Cell Membrane / Q000032:analysis*; D002478:Cells, Cultured; D006023:Glycoproteins / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D006023:Glycoproteins; D008565:Membrane Proteins