Purification and physicochemical characterization of murine T cell replacing factor (TRF).
Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.
D000276:Adjuvants, Immunologic / Q000302:isolation & purification*; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D006133:Growth Substances / Q000032:analysis; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D015847:Interleukin-4; D015848:Interleukin-5; D008222:Lymphokines / Q000032:analysis / Q000302:isolation & purification*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D013601:T-Lymphocytes / Q000276:immunology
D000276:Adjuvants, Immunologic; D006133:Growth Substances; D007075:Immunoglobulin M; D015848:Interleukin-5; D008222:Lymphokines; D015847:Interleukin-4