Affinity purification of an interferon-induced human guanylate-binding protein and its characterization.
Among the proteins that are synthesized only in interferon-treated human cells, a Mr = 67,000 protein has been previously identified by its binding to guanylate agaroses. After a 24-h treatment of human diploid fibroblasts with 200 units/ml of interferon-gamma, about 3 X 10(5) molecules of guanylate-binding protein (GBP) accumulate in each cell. We have developed a one-step purification procedures for GBP using guanylate affinity chromatography. To further elucidate the specific binding of this protein to guanylates, we have used a photoactive probe, 8-azidoguanosine [alpha 32P] triphosphate for the labeling of the GBP. Photolysis of the 8-azido-[alpha-32P]GTP in the presence of GBP results in the covalent attachment of the 32P-guanylate to the GBP. This photolabeling reaction can be inhibited only by guanylates but cannot be inhibited by other nucleotides, suggesting a specific association of GBP to guanylates. Using the purified GBP as an immunogen, we have successfully made rabbit antiserum for GBP. Both the GBP antigen and its guanylate-binding activity are detected only in the cytoplasm of interferon-treated human fibroblasts. The synthesis of the mRNA of GBP is also found in mice exposed to endogenous interferon and in interferon-treated human lymphocytes.
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D006801:Humans; D007106:Immune Sera; D007370:Interferon Type I / Q000494:pharmacology*; D008214:Lymphocytes / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D011817:Rabbits
D007106:Immune Sera; D007370:Interferon Type I; D019204:GTP-Binding Proteins