High-efficiency purification and chemical characterization of B cell stimulatory factor-1/interleukin 4.
B cell stimulatory factor-1/interleukin 4, a lymphokine produced by phorbol ester activated-EL-4 thymoma cells, was purified to homogeneity in good yield by a two-step purification procedure, using affinity chromatography and a single subsequent round of reverse-phase high-performance liquid chromatography. The N-terminal sequence of the first 24 amino acids was consistent with that inferred from the nucleotide sequence of BSF-1 cDNA clones. Amino acid composition analysis also agreed well with that predicted from the nucleotide sequence. A rabbit antibody to a peptide corresponding to positions 100 to 113 inferred from the nucleotide sequence of the cDNA clones bound to BSF-1 purified from EL-4 cells. Purified BSF-1 possesses complex N-linked glycosidic side chains as shown by reduction in Mr from 20,000 to approximately 15,000 by endoglycosidase F but not by endoglycosidase H treatment. Removal of these N-linked sugars does not diminish the activity of BSF-1 as a costimulant in the response of B cells to anti-IgM. By contrast, the reduction of disulfide bonds completely destroyed biologic activity. A monoclonal antibody to BSF-1 blocks its binding to cellular receptors and inhibits biological activities, whereas antibody to the BSF-1 peptide (100-113) has neither effect.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000502:physiology; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006133:Growth Substances / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D015847:Interleukin-4; D008222:Lymphokines / Q000302:isolation & purification* / Q000502:physiology; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006023:Glycoproteins; D006133:Growth Substances; D008222:Lymphokines; D015847:Interleukin-4; D006026:Glycoside Hydrolases