Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes

Journal Article · · Lymphokine Research; (USA)

OSTI ID:6511858

Aiyer, R A; Aggarwal, B B ^[1]

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1. Genentech, Inc., South San Francisco, CA (USA)

High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta.

• OSTI ID: 6511858
• Journal Information: Lymphokine Research; (USA), Journal Name: Lymphokine Research; (USA) Vol. 9:3; ISSN LYRED; ISSN 0277-6766
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
FETAL MEMBRANES
GROWTH FACTORS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MEMBRANE PROTEINS
MEMBRANES
MITOGENS
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLACENTA
PREGNANCY
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
TEMPERATURE DEPENDENCE
TIME DEPENDENCE
TRACER TECHNIQUES