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

R. A. Aiyer, B. B. Aggarwal

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-α) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-α 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-α receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-α 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°C, 24°C and 4°C, respectively. However, the maximum binding obtainable at 4°C was only 40% of that at 37°C. The binding 125I-rhTNF-α to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-α, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-β: previously called lymphotoxin). This is similar to the behavior of TNF-α receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-α and rhTNF-β 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 400,00 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-α are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-β.

Original languageEnglish (US)
Pages (from-to)333-344
Number of pages12
JournalLymphokine research
Volume9
Issue number3
StatePublished - Jan 1 1990

Fingerprint

Tumor Necrosis Factor-alpha
Membranes
Tumor Necrosis Factor Receptors
Lymphotoxin-alpha
Detergents
Placental Extracts
human TNF protein
Ligands
Octoxynol
Cell Extracts
Recombinant Proteins
Radioactivity
Placenta
Gel Chromatography
Binding Sites
Temperature
Proteins

ASJC Scopus subject areas

  • Immunology
  • Hematology

Cite this

Characterization of receptors for recombinant human tumor necrosis factor-α from human placental membranes. / Aiyer, R. A.; Aggarwal, B. B.

In: Lymphokine research, Vol. 9, No. 3, 01.01.1990, p. 333-344.

Research output: Contribution to journalArticle

@article{f44bea6adf6d40f9a38198deb8254299,
title = "Characterization of receptors for recombinant human tumor necrosis factor-α from human placental membranes",
abstract = "High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-α) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-α 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-α receptor could be solubilized by several detergents with optimum extraction being obtained with 1{\%} Triton X-100. The binding of 125I-rhTNF-α 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°C, 24°C and 4°C, respectively. However, the maximum binding obtainable at 4°C was only 40{\%} of that at 37°C. The binding 125I-rhTNF-α to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-α, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-β: previously called lymphotoxin). This is similar to the behavior of TNF-α receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-α and rhTNF-β 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 400,00 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-α are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-β.",
author = "Aiyer, {R. A.} and Aggarwal, {B. B.}",
year = "1990",
month = "1",
day = "1",
language = "English (US)",
volume = "9",
pages = "333--344",
journal = "Journal of Interferon and Cytokine Research",
issn = "1079-9907",
publisher = "Mary Ann Liebert Inc.",
number = "3",

}

TY - JOUR

T1 - Characterization of receptors for recombinant human tumor necrosis factor-α from human placental membranes

AU - Aiyer, R. A.

AU - Aggarwal, B. B.

PY - 1990/1/1

Y1 - 1990/1/1

N2 - High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-α) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-α 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-α receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-α 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°C, 24°C and 4°C, respectively. However, the maximum binding obtainable at 4°C was only 40% of that at 37°C. The binding 125I-rhTNF-α to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-α, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-β: previously called lymphotoxin). This is similar to the behavior of TNF-α receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-α and rhTNF-β 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 400,00 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-α are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-β.

AB - High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-α) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-α 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-α receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-α 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°C, 24°C and 4°C, respectively. However, the maximum binding obtainable at 4°C was only 40% of that at 37°C. The binding 125I-rhTNF-α to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-α, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-β: previously called lymphotoxin). This is similar to the behavior of TNF-α receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-α and rhTNF-β 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 400,00 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-α are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-β.

UR - http://www.scopus.com/inward/record.url?scp=0025353020&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025353020&partnerID=8YFLogxK

M3 - Article

C2 - 2169011

AN - SCOPUS:0025353020

VL - 9

SP - 333

EP - 344

JO - Journal of Interferon and Cytokine Research

JF - Journal of Interferon and Cytokine Research

SN - 1079-9907

IS - 3

ER -