PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “cysteine modification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1998

Effects of chemical modification of cysteines 201 and 317 of band 3 on hemolytic properties of human erythrocytes under hydrostatic pressure.

In the membrane stability of human erythrocytes, the role of two cysteine residues (Cys-201 and Cys-317) in the cytoplasmic domain of band 3 is not clear. So we tried to resolve this problem by examining hemolytic properties under high pressure. From SH contents and spin labeling, it was found that Cys-201 and Cys-317 of band 3 were modified with N-ethylmaleimide (NEM). The hemolysis of intact erythrocytes at 200 MPa was suppressed by the binding of 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), anion transport inhibitor, to band 3. Similarly, the suppressive effect of DIDS was observed in the erythrocyte that Cys-201 and Cys-317 were modified with NEM. These results suggest that the cysteine residues in the cytoplasmic domain of band 3 are not essential for the DIDS-induced membrane stabilization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Peroxynitrite inactivates the human dopamine transporter by modification of cysteine 342: potential mechanism of neurotoxicity in dopamine neurons.

Peroxynitrite (ONOO(-)) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease. ONOO(-) causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO(-) is manifested as a reduction in V(max). Cysteine, dithiothreitol, glutathione, and N-acetyl-cysteine, reagents that interact directly with ONOO(-), prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO(-), whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO(-) the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO(-) treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO(-) reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO(-). Sensitivity to ONOO(-) was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO(-) through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function in vivo (e.g., methamphetamine and MPTP) may exert their effects through ONOO(-)-mediated oxidative stress.

Antioxidants↗
PubMed2000

Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation.

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg(2+) chelatase, such as the ATPase activity of ChlI, and the formation of ChlI-ChlD-MgATP and ChlH-protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg(2+) chelatase activity, and the formation of the ChlI-ChlD-ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI-ChlD-MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg(2+) ions. The simplest explanation for this is that in an intact Mg(2+) chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI-MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

Adenosine Triphosphatases↗
PubMed2001

Structural investigation of the binding of nucleotide to phosphoenolpyruvate carboxykinase by NMR.

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reversible conversion of oxalacetate and GTP to phosphoenolpyruvate (PEP), GDP, and CO2. PEPCK from higher organisms is a monomer, specifically requires GTP or ITP, and uses Mn2+ as the activating cation. Currently, there is no crystal structure of GTP-utilizing PEPCKs. The conformation of the bound nucleotide was determined from transferred nuclear Overhauser effects (trnOe) experiments to determine internuclear proton distances. At 600 MHz in the presence of PEPCK, nOe effects were observed between nucleotide protons. Internuclear distances were calculated from the initial rate of the nOe buildup. These distance constraints were used in energy minimization calculations to determine the conformation of PEPCK-bound GTP. The bound nucleotide has the base oriented anti to the C2'-endo(2E) ribose ring conformation. Relaxation rate studies indicate that there is an additional relaxation effect on the C1' proton upon nucleotide binding to PEPCK. Nucleotide binding to PEPCK-Mn2+ was studied by 1H relaxation rate studies, but results were complicated by long dipole-dipole distances and the presence of competing complexes. Modification of PEPCK by iodoacetamido-TEMPO leads to an inactive enzyme that is spin-labeled at cys273. The interaction of TEMPO-PEPCK with GTP allows for the measurement of nuclear distances between GTP and the spin label. The results suggest that cys273 lies near the ribose ring of the bound nucleotide, but it is too far to be implicated in direct hydrogen bonding interactions consistent with previous results [Makinen, A. L., and Nowak, T. J. Biol. Chem. (1989) 264, 12148], suggesting that cys273 does not actively participate in catalysis. Modification of PEPCK with several cysteine specific modifying agents causes no change in the ability of the enzyme to bind nucleotide as monitored by fluorescence quenching. A correlation between the size of the modifying agent and the maximal observed quenching upon saturation of the enzyme with nucleotide is observed. This suggests a mechanism for inactivation of PEPCK by cysteine modification due to inhibition of a dynamic motion that may occur upon nucleotide binding.

Guanine Nucleotides↗
PubMed2000

The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents.

Membrane permeable N-ethylmaleimide (NEM) and (2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited the rat brain Na(+)-Ca(2+) exchanger RBE-2 (NCX1.5) expressed in HEK 293 cells in a dose dependent manner. 50% inhibition was obtained at 1 mm MTSEA and 1.65 mm NEM. External application of membrane impermeable [2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET) and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not inhibit the transport activity in whole cells. Following reconstitution, however, of RBE-2 transfected cell proteins into proteoliposomes, external application of MTSET and MTSES led to a decrease in transport activity to 42.7 (S.D. = 9.1) and 51% (S.D. = 10.14), respectively. Similar results were obtained also when the rat heart isoform RHE-1 (NCX1.1) or the rat brain isoform RBE-1 (NCX1.4) was expressed. NEM and MTSEA inhibited Na(+) gradient-dependent Ca(2+) uptake also in HEK 293 cells expressing RBE-2/C14A/C20S/ C122S/C780S (numbering corresponds to RBE-2), a mutant in which all putative extracellular cysteines were exchanged. To study the accessibility of different cysteines to covalent modification, surface biotinylation of cells expressing the wild type exchanger and its mutants was carried out using 3-(N-maleimidylpropionyl)biocytin. Surface biotinylation revealed immunoreactive protein derived from the wild type Na(+)-Ca(2+) exchanger only if the transfected cells were exposed to the reducing agent Tris(2-carboxyethyl)phosphine. No reduction was needed when the single cysteine mutants of RBE-2, C14A, C20S, and C780S, were expressed. Treatment of the cells expressing these mutants with MTSET before biotinylation, led to a decrease in the amount of exchanger protein that was revealed. No immunoreactive protein was detected when the quadruple mutant RBE-2, C14A/C20S/C122S/C780S, was biotinylated, suggesting that no additional cysteines are accessible directly from the extracellular face of the membrane. Permeabilizing the cells expressing RBE-2/C14A/C20S/ C122S/C780S with streptolysin O resulted in biotinylation of the exchanger protein. Its amount decreased if exposure to NEM preceded streptolysin O treatment. Our results suggest that Na(+)-Ca(2+) exchange activity is inhibited by covalent modification with sulfhydryl reagents of cysteine residues that are accessible from the cytoplasmic face of the membrane.

Animals↗
PubMed1986

Allosteric regulation of inducer and operator binding to the lactose repressor.

The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range. Inducer binding to the repressor became more cooperative at high pH. The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS). In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation. The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule. All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation. The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein. With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1975

Modification of the cysteine residue of streptococcal dihydrofolate reducatse.

Modification of the single cysteine residue of streptococcal dihydrofolate reducatase with 5,5'-dithiobis (2-nitrobenzoic acid) results in virtually complete inactivation of the enzyme. Reduction of the enzyme-S-nitrobenzoate mixed disulfide with dithiothreitol indicates that 1.0 cysteine has been modified and approximately 70 percent of the original enzyme activity restored. Circular dichroic and fluorescence studies suggest that a localized conformational change involving aromatic residues, possibly tryptophan, has occurred following modification. In addition, nearly stoichiometric amounts of p-hydroxymercuribenzoate lead to complete inactivation of the reducatas. It is suggested that the S-nitrobenzoate moiety bound to the cysteine residue of the enzyme may perturb one or more aromatic side chains and lead to a distortion of the hydrophobic substrate binding site.

Benzoates↗
PubMed1975

2,3-Dioxo-5-indolinesulfonic acid, a new highly specific reagent for modification of tryptophan in peptides and proteins.

A new reagent, 2,3-dioxo-5-indolinesulfonic acid (DISA), has been investigated for its specificity to react with tryptophan and tryptophan residues in peptides and proteins. On reaction of 19 amino acids with 1 M excess of DISA in 0.1 M acetic acid (pH 2.9), considerable (50%) modification of tryptophan was obtained within 50 min and no other amino acid was modified. After reaction for 5 h, only proline showed very slight (7%) modification. On reaction in the presence of 5 M excess of DISA, tryptophan was very rapidly modified. Modification of proline became appreciable in the presence of this molar excess of reagent and cysteine modification, although much smaller, became detectable (12 h, 7%). However, proline modification was completely prevented when this amino acid was engaged in a peptide linkage, even after reaction for 45 h in the presence of 5 or 10 M excess of DISA per proline residue. Reaction of egg albumin with 50 M excess of DISA was entirely specific for tryptophan and showed no modification of proline or cysteine residues. The reagent offers the advantages of stability, easy handling, high water solubility and high specificity. It affords protein and peptide derivatives that are completely water soluble because of the polar nature of the added group. The yellow color (lambda max, 367 nm) of the derivatives offers advantages of easy determination of the extent and location of the modification.

Amino Acids↗
PubMed1979

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.

Amino Acids↗
PubMed2001

Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion products. Based on these results, we propose that cigarette smoke may contribute to atherogenesis by inhibiting the activities of plasma PAF-AH and LCAT, but the nature of this inhibition differs for the enzymes.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2005

Aminoethylation in model peptides reveals conditions for maximizing thiol specificity.

Control of pH in aminoethylation reactions is critical for maintaining high selectivity towards cysteine modification. Measurement of aminoethylation rate constants by liquid chromatography mass spectrometry demonstrates reaction selectivity of cysteine>>amino-terminus>>histidine. Lysine and methionine were not reactive at the conditions used. For thiol modification, the acid/base property of the gamma-thialysine residue measured by NMR results in a 1.15 decrease in pK(a) (relative to a lysine residue). NMR confirms ethylene imine is the reactive intermediate for alkylation of peptide nucleophiles with bromoethylamine. Conversion of bromoethylamine into ethylene imine prior to exposure to the target thiol, provides a reagent that promotes selectivity by allowing precise control of reaction pH. Reaction selectivity plots of relative aminoethylation rates for cysteine, histidine, and N-terminus imine demonstrate increasing alkaline conditions favors thiol modification. When applied to protein modification, the conversion of bromoethylamine into ethylene imine and buffering at alkaline pH will allow optimal cysteine residue aminoethylation.

Amino Acids↗
PubMed2007

Proteomic identification of S-nitrosylated proteins in endothelial cells.

Nitric oxide (NO) produced in endothelial cells exerts important roles in the vascular system. In recent years, posttranslational modifications induced by NO have been increasingly studied and, among them, cysteine modification by S-nitrosylation (also called S-nitrosation) has been hypothesized to represent a relevant mechanism for cell signaling. Thus, knowledge of the proteins that can be S-nitrosylated in endothelial cells will help to better understand the possible role of this modification. We describe a protocol to identify the S-nitrosylome or S-nitrosoproteome of endothelial cells, based on the specific derivatization of the S-nitrosylation, substituting it by a biotinylation, and the purification of the biotinylated proteins.

Cysteine↗
PubMed1999

The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue.

The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of neurotransmitter transporters that maintain low synaptic levels of the transmitter. Transport by GAT-1 is sensitive to the polar sulfhydryl reagent 2-aminoethyl methanethiosulfonate. Following replacement of endogenous cysteines to other residues by site-directed mutagenesis, we have identified cysteine 399 as the major determinant of the sensitivity of the transporter to sulfhydryl modification. Cysteine-399 is located in the intracellular loop connecting putative transmembrane domains eight and nine. Binding of both sodium and chloride leads to a reduced sensitivity to sulfhydryl reagents, whereas subsequent binding of GABA increases it. Strikingly binding of the nontransportable GABA analogue SKF100330A gives rise to a marked protection against sulfhydryl modification. These effects were not observed in C399S transporters. Under standard conditions GAT-1 is almost insensitive toward the impermeant 2-(trimethylammonium)ethyl methanethiosulfonate. However, in a chloride-free medium, addition of SKF100330A renders wild type GAT-1, but not C399S, very sensitive to this impermeant reagent. These observations indicate that the accessibility of cysteine 399 is highly dependent on the conformation of GAT-1. Consequently, topological assignments based on accessibility of endogeneous or engineered cysteines to small polar sulfhydryl reagents need to be interpreted with extreme caution.

Carrier Proteins↗
PubMed2000

Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S.

lambda S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including lambda S and 21 S, have the "dual-start" regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic "helical-hairpin" topology, with N and C termini on the cytoplasmic side of the membrane. However, lambda S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of lambda S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin S(lambda) has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.

Amino Acid Sequence↗