Purification and sequencing of glycosylation variants of BSF-1, as a MAF, from the EL-4 leukaemia cell line.
Macrophage activation activity was characterized from a PMA-induced subclone of the murine EL-4 leukaemic cell line. The MAF was purified from the cell line culture supernatant by concentration, CM-Sepharose and lentil lectin Sepharose chromatography, AcA 54 gel filtration, Mono Q FPLC and reverse phase HPLC. Four protein bands of different abundance were observed on SDS-PAGE with molecular weights of 17,500 to 21,000 Da. Three of the four proteins were sequenced from the N-terminal and shared homology with the published sequence of BSF-1. Variation of the molecular weight due to glycosylation was demonstrated by N-glycanase treatment, all four proteins gave a band of 14,200 Da after deglycosylation. Both glycosylated and deglycosylated forms of BSF-1 were equally active in the MAF assay. A monoclonal antibody to BSF-1 neutralized 80% of the activity from crude culture supernatants in the MAF assay. These studies have indicated that BSF-1 is the major, if not the only, MAF activity from this particular subline of the murine EL-4 leukaemic cell line.
Sutton
C
C
Celltech Limited, Berkshire, UK.
Depledge
P
P
Bawden
L
L
Carne
A
A
Meltzer
M
M
Newton
V
V
Vodinelich
L
L
D000595:Amino Acid Sequence; D000818:Animals; D006031:Glycosylation; D015847:Interleukin-4; D007378:Interleukins / Q000302:isolation & purification*; D004915:Leukemia, Erythroblastic, Acute / Q000276:immunology*; D008222:Lymphokines / Q000302:isolation & purification*; D016215:Macrophage-Activating Factors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000276:immunology
D007378:Interleukins; D008222:Lymphokines; D016215:Macrophage-Activating Factors; D015847:Interleukin-4; D013755:Tetradecanoylphorbol Acetate