Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization.
Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.
D002846:Chromatography, Affinity; D003208:Concanavalin A; D000939:Epitopes; D006801:Humans; D007118:Immunoassay; D007372:Interferons / Q000302:isolation & purification* / Q000494:pharmacology; D007962:Leukocytes / Q000032:analysis*; D008970:Molecular Weight; D009522:Newcastle disease virus / Q000187:drug effects; D013045:Species Specificity; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
D000939:Epitopes; D003208:Concanavalin A; D007372:Interferons