Purification of type I and type II tumor necrosis factor receptors from human lung tissue.
Two receptors for tumor necrosis factor-alpha (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of 75 and 55 kD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-kD receptors could be precipitated from 125I-surface-labeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.
Abdolrasulnia
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Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212.
Shepherd
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VL
D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008168:Lung / Q000737:chemistry* / Q000276:immunology; D011233:Precipitin Tests; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha