Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial characterization.
The receptor for human tumor necrosis factor-alpha (TNF-alpha) was isolated from a subclone of the human histiocytic lymphoma cell line U937. These cells exhibit a single class of high affinity receptors (Kd = 0.51 +/- 0.25 nM) with an average density of 55,000 +/- 5,000 binding sites/cell. After solubilization with detergent, the receptor retained its ability to bind free TNF-alpha but failed to bind to TNF-alpha immobilized on various solid supports. For receptor purification, 125I-TNF-alpha was covalently attached to the receptor on intact cells by the bifunctional cross-linking reagents ethylene glycolbis(succinimidylsuccinate) or 3,3-dithiobis(sulfosuccinimidylpropionate). The cells were then solubilized with the nonionic detergent Triton X-100, and the supernatants, clarified by centrifugation, were passed over an IgG-Sepharose column prepared from TNF-alpha antiserum. The receptor-rich fraction from the antibody column was further purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These two steps together provided approximately 165,000-fold purification of the TNF-alpha receptor. The TNF-alpha receptor-ligand complex obtained by this method had a subunit molecular weight of 100,000 +/- 5,000 when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but on gel filtration the complex migrated with an apparent molecular weight of 480,000 +/- 32,000. However, the receptor showed a molecular weight of 65,000 +/- 32,000 when gel filtration was performed in the absence of ligand. Additional characteristics of the receptor are discussed.
Stauber
G B
GB
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
Aiyer
R A
RA
Aggarwal
B B
BB
D001667:Binding, Competitive; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018124:Receptors, Tumor Necrosis Factor; D012995:Solubility
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor