Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.
Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.
Hayakawa
M
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Hori
T
T
Shibamoto
S
S
Tsujimoto
M
M
Oku
N
N
Ito
F
F
D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins