PMID- 10699356
OWN - NLM
STAT- MEDLINE
DCOM- 20000620
LR  - 20190915
IS  - 0887-2333 (Print)
IS  - 0887-2333 (Linking)
VI  - 14
IP  - 1
DP  - 2000 Feb
TI  - Inhibition of NF-kappaB-DNA binding by mercuric ion: utility of the non-thiol 
      reductant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), on detection of 
      impaired NF-kappaB-DNA binding by thiol-directed agents.
PG  - 7-16
AB  - Mercuric ion (Hg(2+)), a potent thiol inhibitor, prevents expression of nuclear 
      factor kappaB (NF-kappaB) by mercaptide bond formation with a critical cysteine 
      moiety (cys(62)) on the p50 subunit required for DNA binding. NF-kappaB-DNA 
      binding is typically measured in reaction mixtures in which dithiothreitol (DTT) 
      or other thiol reductants are used to maintain cys(62) in the reduced state. 
      However, the presence of thiol reductants prevents accurate assessment of the 
      Hg(2+) concentration required to prevent NF-kappaB-DNA binding because of 
      competitive mercaptide bond formation. In the present studies we evaluated the 
      efficacy of tris(2-carboxyethyl)phosphine-HCl (TCEP), a non-thiol reducing agent 
      which does not bind Hg(2+), on NF-kappaB-DNA binding in vitro, using recombinant 
      p50 protein and a (32)P-labelled kappaB oligonucleotide. We also measured the 
      minimal Hg(2+) concentration required to prevent this interaction in the presence 
      of either reagent. DTT promoted NF-kappaB-DNA binding in concentrations from 0.25 
      to 2.6mM in binding reactions. However, in the presence of 0.25mM DTT, inhibition 
      of NF-kappaB binding was seen only at Hg(2+) concentrations greater than 100 
      microM and results were highly variable. In contrast, TCEP promoted NF-kappaB-DNA 
      binding in a dose-related manner in concentrations from 0.25 to 6mM. In the 
      presence of even 6mM TCEP, Hg(2+) prevented NF-kappaB-DNA binding at 
      concentrations as low as 20 microM in binding reactions. Similar findings were 
      observed with regard to the thiol alkylating agent N-ethylmaleimide (NEM). These 
      findings demonstrate the utility of TCEP as reductant in nuclear binding reaction 
      assays involving the interaction of thiol constituents. They also demonstrate 
      inhibition of NF-kappaB-DNA binding at Hg(2+) concentrations comparable to those 
      known to initiate toxicity and apoptotic cell death in vivo.
FAU - Dieguez-Acuna, F J
AU  - Dieguez-Acuna FJ
AD  - Department of Environmental Health, 4225 Roosevelt Way NE, Suite 100, University 
      of Washington, Seattle, WA 98105, USA.
FAU - Woods, J S
AU  - Woods JS
LA  - eng
GR  - ES04696/ES/NIEHS NIH HHS/United States
GR  - P30ES07033/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Toxicol In Vitro
JT  - Toxicology in vitro : an international journal published in association with 
      BIBRA
JID - 8712158
RN  - 0 (NF-kappa B)
RN  - 0 (Oligonucleotides)
RN  - 0 (Phosphines)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Reducing Agents)
RN  - 0 (Sulfhydryl Reagents)
RN  - 22OAC2MO2S (tris(2-carboxyethyl)phosphine)
RN  - 9007-49-2 (DNA)
RN  - FXS1BY2PGL (Mercury)
RN  - O3C74ACM9V (Ethylmaleimide)
RN  - T8ID5YZU6Y (Dithiothreitol)
SB  - IM
MH  - DNA/*drug effects/metabolism
MH  - Dithiothreitol/pharmacology
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Ethylmaleimide/pharmacology
MH  - Humans
MH  - Mercury/*pharmacology
MH  - NF-kappa B/*antagonists & inhibitors/drug effects/metabolism
MH  - Oligonucleotides/pharmacology
MH  - Phosphines/*pharmacology
MH  - Recombinant Proteins/pharmacology
MH  - Reducing Agents/*pharmacology
MH  - Sulfhydryl Reagents/*pharmacology
EDAT- 2000/03/04 09:00
MHDA- 2000/06/24 11:00
CRDT- 2000/03/04 09:00
PHST- 2000/03/04 09:00 [pubmed]
PHST- 2000/06/24 11:00 [medline]
PHST- 2000/03/04 09:00 [entrez]
AID - S0887-2333(99)00091-0 [pii]
AID - 10.1016/s0887-2333(99)00091-0 [doi]
PST - ppublish
SO  - Toxicol In Vitro. 2000 Feb;14(1):7-16. doi: 10.1016/s0887-2333(99)00091-0.