Characterization of suppressive immunoglobulin-binding factor (IBF). II. Purification and molecular weight determination of IBF produced by L-5178-Y theta-positive lymphoma

Abstract

L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes. IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d. These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,00 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.