What we can learn from the effects of thiol reagents on transport proteins

Iwaarden, Pierre R. van; Driessen, Arnold J. M.; Konings, Wil N.

doi:10.1016/0304-4157(92)90037-b

Cited by 105 publications

(62 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…HgCl 2 and methylmercury are the most common forms of mercury to which humans are exposed [4,[6][7]; mersalyl has been used as a prototypal hydrophilic mercury compound which is frequently used as a specific reagent for Cys residues of proteins [35]. Preliminary studies performed with hydrophilic SH reagents, suggested that the reconstituted transporter contains thiol groups of cysteine exposed towards the extraliposomal compartment, which corresponds to the extracellular environment; these data correlated well with the predicted hydropathy profile of ASCT2 [27,40].…”

Section: Discussionmentioning

confidence: 99%

“…3). The involvement of the CXXC motif in the inhibition is in agreement with the higher affinity of the transporter for HgCl 2 (Hg 2+ ), which can react with two vicinal SH, than to methylmercury and mersalyl, which can react with only one Cys [35]. Among other metal cations which are known to react with vicinal SH groups [35], Cu 2+ strongly inhibited the reconstituted transporter (IC 50 , 20 µM) with a non competitive mechanism; whereas Cd 2+ and Zn 2+ were poor inhibitors with IC 50 > 500 µM (Oppedisano F. and Indiveri C. unpoublished…”

Section: Discussionmentioning

confidence: 99%

“…It is known that mercuric compounds react with Cys residues of proteins by Hg-S covalent bond [35]. To verify if the mercuric reagents reacted with Cys residues of the glutamine/amino acid transporter, the effect of DTE, which reverses the Hg-S linkage, was tested.…”

Section: Inhibitionmentioning

confidence: 99%

See 2 more Smart Citations

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Oppedisano

Galluccio

Indiveri

2010

Biochemical Pharmacology

View full text Add to dashboard Cite

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Oppedisano

Galluccio

Indiveri

2010

Biochemical Pharmacology

View full text Add to dashboard Cite

show abstract

“…Finally, unlike the complicated protective effect of substrates on NEM modification, the stimulatory effect of TPP ϩ is more comprehensible and implies widespread conformational changes that occur upon binding of TPP ϩ to MdfA (26 -28). However, one cannot rule out the influence of the positive charge of TPP ϩ on the de-protonation of certain nearby Cys residues, which might enhance their reactivity with NEM (35).…”

Section: Effect Of the Substrates On [ 14 C]nem Labeling Of Single-mentioning

confidence: 99%

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Adler

Bibi

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (⌬˜H؉, inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.

show abstract

“…Oxidation of SH groups in proteins by disulfides such as DTN B is specific for cysteine residues and results in the formation of mixed disulfides (Van Iwaarden et al, 1992). GST-TPH-CC was exposed to varying concentrations of DTN B in the absence of DTT at 30°C for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of the Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Attack on Critical Sulfhydryls that Spare the Enzyme Iron Center

Kuhn

Arthur²

1997

J. Neurosci.

View full text Add to dashboard Cite

Tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT), is irreversibly inactivated by nitric oxide (NO). We have expressed brain TPH as a recombinant glutathione-Stransferase fusion protein and delineated the catalytic domain of the enzyme as the region spanning amino acids 99-444. Highly purified TPH catalytic core, like the native enzyme from brain, is inactivated by NO in a concentration-dependent manner. Removal of iron from TPH produces an apoenzyme with low activity that can be reconverted to its highly active holoform by the addition of ferrous iron. Apo-TPH exposed to NO cannot be reactivated by iron. Treatment of holo-TPH (ironloaded) with the disulfide 5,5Ј-dithio-bis (2-nitrobenzoic acid) (DTNB) causes an inactivation of TPH that is readily reversed by dithiothreitol (DTT). DTNB-treated TPH [sulfhydryl (SH)-protected] exposed to NO is returned to full activity by thiol reduction with DTT. The inactivation of native TPH by NO cannot be reversed by either iron or DTT. These data indicate that NO inactivates TPH by selective action on critical SH groups (i.e., cysteine residues) while sparing catalytic iron sites within the enzyme. The results are interpreted with reference to the substituted amphetamines, which are neurotoxic to 5-HT neurons, that inactivate TPH in vivo and are now known to produce NO and other reactive oxygen species in vivo. Key words: tryptophan hydroxylase; nitric oxide; sulfhydryls; catalytic iron site; serotonin; neurotoxic amphetaminesTryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] (TPH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) (Jequier et al., 1967). The synthesis of 5-HT can proceed only through this enzymecatalyzed step. The activity of TPH controls the amount of 5-HT produced and released from neurons (Gartside et al., 1992;Oluyomi et al., 1994), indicating that this enzyme regulates not just 5-HT synthesis but its f unction as well. As a neurotransmitter, 5-HT mediates pain, sleep, thermoregulation, and food intake. From a clinical perspective, altered 5-HT f unction has been implicated in depression, obsessive-compulsive disorder, autism, and impulsive self-destructive behaviors such as aggression, suicide, and drug taking (Schatzberg and Nemeroff, 1995).Selected amphetamines have profound effects on the 5-HT neuronal system. Methylenedioxymethamphetamine (ecstasy) (MDM A) and p-chloroamphetamine cause extensive destruction of 5-HT neurons. An early manifestation of their effects is a significant inactivation of TPH (for reviews, see Gibb et al., 1993;Steele et al., 1994;Seiden and Sabol, 1996). The mechanisms underlying these effects on TPH are not known, but emerging data implicate drug-induced production of reactive oxygen species (ROS) and nitric oxide (NO). The cellular effects of ROS or NO cannot be predicted a priori: NO can be toxic to some cells (Lipton et al., 1993;Da...

show abstract

What we can learn from the effects of thiol reagents on transport proteins

Cited by 105 publications

References 68 publications

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Molecular Mechanism of the Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Attack on Critical Sulfhydryls that Spare the Enzyme Iron Center

Contact Info

Product

Resources

About