Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts
Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.
- Research Organization:
- Genentech, Inc., South San Francisco, CA
- OSTI ID:
- 6045634
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 22
- Country of Publication:
- United States
- Language:
- English
Similar Records
Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes
Purification and characterization of an inhibitor (soluble tumor necrosis factor receptor) for tumor necrosis factor and lymphotoxin obtained from the serum ultrafiltrates of human cancer patients
Related Subjects
FIBROBLASTS
CONFIGURATION INTERACTION
LYSIS
TOXINS
TRITIUM COMPOUNDS
KINETICS
L CELLS
MICE
RECEPTORS
ANIMAL CELLS
ANIMALS
ANTIGENS
CONNECTIVE TISSUE CELLS
LABELLED COMPOUNDS
MAMMALS
MATERIALS
RODENTS
SOMATIC CELLS
TOXIC MATERIALS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques