Effects of Dithiothreitol on Protein Activity Unrelated to Thiol–Disulfide Exchange: For Consideration in the Analysis of Protein Function with Cleland's Reagent

Alliegro, Mark C.

doi:10.1006/abio.2000.4557

Cited by 63 publications

(48 citation statements)

References 14 publications

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“…Therefore, one would expect that DTT, once passed through the cell membrane, should not affect protein sulfhydryls on the intracellular side. The remaining sensitivity of 3CYS to DTT observed in our experiments could thus be unrelated to thiol-disulfide exchange (Alliegro, 2000).…”

Section: Discussionmentioning

confidence: 65%

“…Note shift in V 1/2 toward more positive potentials in 3CYS mutant. at ASPET Journals on May 9, 2018 molpharm.aspetjournals.org Downloaded from maining "nonspecific" effects unrelated to thiol-disulfide exchange at TRPV1 (Alliegro, 2000). Previous studies on NR1/ NR2A (Choi et al, 2001) and GABA A (Wilkins and Smart, 2002) receptors demonstrated that the reversible effects of DTT might be caused by the chelation of trace amounts of divalent cations rather than by redox-based mechanisms.…”

Section: Resultsmentioning

confidence: 99%

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Reducing and Oxidizing Agents Sensitize Heat-Activated Vanilloid Receptor (TRPV1) Current

Sušánková

Tousova²,

Vyklický³

et al. 2006

Mol Pharmacol

101

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We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membranepermeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 M) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5Ј-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membraneimpermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.The capsaicin receptor transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific ion channel that plays an important role in thermal nociception and inflammatory hyperalgesia (Caterina et al., 1997). This channel can be activated by vanilloid compounds, low pH, and noxious heat, and its function is modulated by a wide range of endogenous and exogenous agents (for review, see Planells-Cases et al., 2005). Among them, redox-active substances have recently been shown to modulate TRPV1 receptor activity (Vyklicky et al., 2002;Jin et al., 2004); however, it is not yet clear whether these effects are mediated through the direct modification of sulfhydryl groups present in the TRPV1 channel protein complex. As demonstrated by earlier radioligand binding studies, both the reducing and oxidizing agents diminished the apparent affinity and positive cooperativity characteristic of [ 3 H]resiniferatoxin binding . From these studies, it has become apparent that cooperation between the TRPV1 subunits is, at least in part, subject to redox modulation. Also, our earlier studies demonstrated that the sulfhydryl redox agent dithiothreitol (DTT) strongly potentiates both the native and recombinant rat TRPV1 channel when applied at millimolar concentrations to the external solution under intact whole...

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Section: Discussionmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Reducing and Oxidizing Agents Sensitize Heat-Activated Vanilloid Receptor (TRPV1) Current

Sušánková

Tousova²,

Vyklický³

et al. 2006

Mol Pharmacol

101

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“…However, the mechanism of this inactivation is not as yet understood. The inactivation may not be simply due to disulfide reduction, as effects of DTT on protein activity unrelated to disulfide reduction have been reported (22). It has also been shown that ASM can be activated in terms of its enzymatic activity, such as by lysosomal lipids and the sphingolipid activator protein SAP-C (23).…”

Section: Acid Sphingomyelinase (Asm)mentioning

confidence: 99%

Activation of Human Acid Sphingomyelinase through Modification or Deletion of C-terminal Cysteine

Qiu¹,

Edmunds²,

Baker-Malcolm³

et al. 2003

Journal of Biological Chemistry

131

119

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One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at ؊20°C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys 629 ). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by Cterminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

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“…In many cases, retention of activity is dependent upon the presence of a strong sulfhydryl reagent, such as 2-mercaptoethanol (2-Me) or dithiothreitol, to maintain essential thiol/ disulfide bonds. 1,2 Because such critical structural conformations are essential for function, investigations were undertaken to determine what effect sulfhydryl molecules might impart on immune cell reactivities in tissue culture under a non-physiological, oxygen environment. We first tested, if the then, Mishell/ Dutton protocol 3 for in vitro primary murine antibody synthesis at physiological levels of oxygen, could be replaced by addition of 2-Me to cultures that were incubated in air.…”

Section: Introductionmentioning

confidence: 99%

Obesity, longevity, quality of life

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Effects of Dithiothreitol on Protein Activity Unrelated to Thiol–Disulfide Exchange: For Consideration in the Analysis of Protein Function with Cleland's Reagent

Cited by 63 publications

References 14 publications

Reducing and Oxidizing Agents Sensitize Heat-Activated Vanilloid Receptor (TRPV1) Current

Reducing and Oxidizing Agents Sensitize Heat-Activated Vanilloid Receptor (TRPV1) Current

Activation of Human Acid Sphingomyelinase through Modification or Deletion of C-terminal Cysteine

Obesity, longevity, quality of life

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