Biochemical characterization of murine glycosylation-inhibiting factor.
The glycosylation-inhibiting factor (GIF) was isolated from serum-free culture supernatants of the murine T-cell hybridoma, 231F1 cells, by using an immunosorbent coupled with the monoclonal anti-lipomodulin antibody. The isolated lymphokine is a 14-kDa protein with a pI of 5.5, as determined by SDS/PAGE and two-dimensional gel electrophoresis. Fractionation of a mixture of radiolabeled GIF with culture supernatant of the 231F1 cells on ion-exchange and reverse-phase columns and by gel filtration demonstrated homogeneity of the 14-kDa GIF and confirmed that the bioactivity of GIF and the antigenic determinant recognized by the monoclonal anti-GIF antibody are associated with the 14-kDa protein. The 125I-labeled 14-kDa protein binds to the murine T-cell hybridoma 12H5 cells, which have been used for bioassay of GIF, and the murine B-cell line A20.3 cells, but the binding of the protein to resting murine splenic lymphocytes was barely detectable. Under the same experimental conditions, binding of the 125I-labeled recombinant human lipocortin I to the 12H5 cells was not detectable. In contrast, the 125I-labeled lipocortin, but not the 14-kDa GIF, bound to phosphatidylserine vesicles. The results indicate that GIF does not belong to the anexin family.
La Jolla Institute for Allergy and Immunology, CA 92037.
Y
Y
Tagaya
A
A
Mori
K
K
Ishizaka
D000818:Animals; D017302:Annexins; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000276:immunology; D006031:Glycosylation; D006825:Hybridomas / Q000276:immunology; D008222:Lymphokines / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D029584:Prostatic Secretory Proteins; D013601:T-Lymphocytes / Q000276:immunology*
D017302:Annexins; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D008222:Lymphokines; D029584:Prostatic Secretory Proteins; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; C030958:lipomodulin