Purification and characterization of the lymphocyte function-associated-2 (LFA-2) molecule.
The lymphocyte function-associated-2 (LFA-2) molecule, equivalent to CD2 and the E rosette receptor, was purified by MAb affinity chromatography from the Jurkat T lymphoma cell line. Jurkat was selected for its high level of expression of 1.0 X 10(5) sites/cell. A two-site radioimmunometric assay was developed to monitor purification. From 50 g of packed cells, 230 micrograms of LFA-2 was obtained with 65% yield of antigenic activity with a purification factor of 13,000. A major component of 58,000 and 54,000 was obtained that corresponded to LFA-2 antigenic activity as shown by immunoblotting and immunoprecipitation. The doublet was resolved by 2D IEF-SDS-PAGE into components of pI = 5.5 and 5.6. Smaller amounts of lower Mr components were also seen. All these components appeared related by processing or proteolytic breakdown, as shown by Cleveland peptide mapping. The LFA-2 deoxycholate complex had an apparent Mr of 68,000 by gel filtration, suggesting it was monomeric. Purified LFA-2 inhibited rosetting of T lymphocytes with sheep E, and addition to preformed rosettes caused their disruption. Inhibitory activity was absorbed by sheep E. This is the first evidence that the CD2/LFA-2 molecule can directly bind to sheep E. Purified LFA-2 should be useful for the further biochemical and functional characterization of this molecule.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D007525:Isoelectric Focusing; D008077:Lipoproteins, LDL / Q000302:isolation & purification; D016169:Lymphocyte Function-Associated Antigen-1; D008223:Lymphoma / Q000276:immunology; D011233:Precipitin Tests; D012397:Rosette Formation; D012756:Sheep; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008077:Lipoproteins, LDL; D016169:Lymphocyte Function-Associated Antigen-1; C022060:rosette-inhibiting factor