Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab′)₂ fragments had been coupled. The intact IgG column bound 35.7% of the applied counts, whereas the F(ab′)₂ columns bound 2.8%. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000, 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same subclasses of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.     

Isolation of a murine leukemia FC receptor by selective release induced by surface redistribution.

A protein which binds to the Fc region of IgG has been isolated from the murine leukemia L1210. The isolation technique involves surface cross-linking of the cells's Fc receptors with the use of aggregated human IgG and anti-human IgG. This results in the redistribution (patch formation and capping) of the cells's Fc receptors. Lactoperoxidase-catalyzed radioiodination of the cells before complex binding indicates that Fc receptor redistribution results in the selective release of surface proteins. SDS-PAGE analyses of the supernatants from cells thus treated reveals a major peak corresponding to a molecular weight of 45,000 daltons. This protein has been purified from the cell supernatants by immunoprecipitation and chromatography of the percipitates on Sephadex G-200 under dissociating conditions. After separation from the immune complex this protein can be bound to heat-aggregated IgG, but not aggregated F(ab')2 fragments. The 45,000 dalton protein appears to be the Fc receptor which has been released from the cell surface in association with the complex.