Binding of 125I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells.
We investigated the binding of 125I-labeled beta interferon (IFN-beta Ser17), a nonglycosylated recombinant human fibroblast interferon in which cysteine at position 17 is replaced by serine by site-specific mutagenesis. An optimized chloramine T radiolabeling method produced a highly labeled, fully active 125I-IFN suitable for these studies. Unlike the case with the chloramine T method, incorporation of a single mole of Bolton-Hunter reagent into a mole of IFN-beta Ser17 led to nearly complete loss of biological activity. 125I-IFN-beta Ser17, prepared by the chloramine T method, bound specifically to human lymphoblastoid cells (Daudi) with a dissociation constant of 0.24 nM. The number of binding sites per cell was 4,000. In competition assays, unlabeled beta interferons (native, recombinant IFN-beta Cys17, and various preparations of IFN-beta Ser17) equally displaced labeled IFN-beta Ser17 on Daudi cells. Recombinant IFN-alpha-1 displaced 125I-IFN-beta binding to Daudi cells less efficiently than did unlabeled native or recombinant beta interferon. However, at the concentrations tested, native gamma interferon showed no competition with 125I-IFN. Our results indicate that IFN-beta Ser17 and native IFN-beta posses similar binding properties.
D001665:Binding Sites; D001667:Binding, Competitive; D002700:Chloramines / Q000378:metabolism; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism*; D000068556:Interferon beta-1a; D000068576:Interferon beta-1b; D016899:Interferon-beta; D007371:Interferon-gamma / Q000378:metabolism; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D013388:Succinimides; D014105:Tosyl Compounds
D002700:Chloramines; D007370:Interferon Type I; D007457:Iodine Radioisotopes; D013388:Succinimides; D014105:Tosyl Compounds; C016300:chloramine-T; D000068576:Interferon beta-1b; C022581:Bolton-Hunter reagent; D016899:Interferon-beta; D007371:Interferon-gamma; D000068556:Interferon beta-1a