Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase.
AuthorsBhattacharjee H,Rosen BP
JournalThe Journal of biological chemistry
PubMed ID8798705
A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.
AuthorsRatner V,Kahana E,Eichler M,Haas E
JournalBioconjugate chemistry
PubMed ID12236801
Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state ... More
Reactivity of mitochondrial sulfhydryl groups toward dithionitrobenzoic acid and bromobimanes under oligomycin-inhibited and uncoupling conditions.
AuthorsFreisleben HJ, Fuchs J, Mainka L, Zimmer G
JournalArch Biochem Biophys
PubMed ID2845867
Thiol reactivity was determined in rat heart mitochondria using chromophores of differing polarities: monobromobimane (MB), dithionitrobenzoate (NbS2), and bromobimane-q (MQ). The purpose of this study is to correlate reaction rates of protein thiols in the mitochondrial membrane with the oligomycin-inhibited and uncoupled states: In all cases investigated the reactivity of ... More
Sulfhydryl groups of rabbit liver arylsulfatase A.
AuthorsWaheed A, Van Etten RL
JournalBiochim Biophys Acta
PubMed ID2864083
Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan ... More
Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton.
'Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of ... More
Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein.
AuthorsJain S, McGinnes LW, Morrison TG
JournalJ Virol
PubMed ID17151113
'Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought ... More
Reassessment of Ellman's reagent.
AuthorsRiddles PW, Blakeley RL, Zerner B
JournalMethods Enzymol
PubMed ID6855597
Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin.
AuthorsGraceffa P
JournalBiochemistry
PubMed ID9092808
'Smooth muscle caldesmon is a single polypeptide chain with its NH2- and COOH-terminal domains separated by a long alpha-helix. Caldesmon was labeled at either Cys-153 in the NH2 domain or Cys-580 in the COOH domain with a variety of fluorescence probes. Fluorescence intensity, peak position, and polarization of probes on ... More
Evaluation of methods for measuring cellular glutathione content using flow cytometry.
AuthorsHedley DW, Chow S
JournalCytometry
PubMed ID8026225
'The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity ... More
Active site titration of the tyrosine phosphatases SHP-1 and PTP1B using aromatic disulfides. Reaction with the essential cysteine residue in the active site.
AuthorsPregel MJ, Storer AC
JournalJ Biol Chem
PubMed ID9295292
'Aromatic disulfides were found to inactivate truncated forms of the SHP-1 and PTP1B phosphatases by reaction with the essential active site cysteine residue. For truncated SHP-1 at pH 5.0, the reaction proceeded through an initial burst phase followed by a slower secondary phase. Our experiments demonstrated that the burst phase ... More
Intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy.
AuthorsNeyroz P, Zambelli B, Ciurli S
JournalBiochemistry
PubMed ID16846235
'UreG is an essential protein for the in vivo activation of urease. In a previous study, UreG from Bacillus pasteurii was shown to behave as an intrinsically unstructured dimeric protein. Here, intrinsic and extrinsic fluorescence experiments were performed, in the absence and presence of denaturant, to provide information about the ... More
AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC.
AuthorsPoole LB, Godzik A, Nayeem A, Schmitt JD
JournalBiochemistry
PubMed ID10828978
'AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates. The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) ... More
Properties of spin and fluorescent labels at a receptor-ligand interface.
AuthorsOwenius R, Osterlund M, Lindgren M, Svensson M, Olsen OH, Persson E, Freskgård PO, Carlsson U
JournalBiophys J
PubMed ID10512843
'Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently ... More
Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.
AuthorsDe La Cruz EM, Pollard TD
JournalBiochemistry
PubMed ID7727403
'We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The ... More
Sulfhydryl-modifying reagents reversibly inhibit binding of glucocorticoid-receptor complexes to DNA-cellulose.
AuthorsBodwell JE, Holbrook NJ, Munck A
JournalBiochemistry
PubMed ID6722099
'Glucocorticoid -receptor complexes from intact rat thymus cells incubated with [3H]dexamethasone at 0 degree C are in the nonactivated form and do not bind to DNA-cellulose. Upon being warmed, they are transformed to activated complexes that bind to DNA-cellulose at 0 degree C. We have found that treatment of dexamethasone-receptor ... More
Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin.
AuthorsGraceffa P
JournalJ Biol Chem
PubMed ID8530428
'The NH2- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5''-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310). Upon ... More
A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein.
AuthorsOkoh MP, Hunter JL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID17144669
'A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they ... More
Characterization of recombinant yeast dolichyl mannosyl phosphate synthase and site-directed mutagenesis of its cysteine residues.
AuthorsForsee WT, McPherson D, Schutzbach JS
JournalEur J Biochem
PubMed ID9108271
'Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and ... More
Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli.
AuthorsGehring AM, Lees WJ, Mindiola DJ, Walsh CT, Brown ED
JournalBiochemistry
PubMed ID8555230
'The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788-5795], a fundamental precursor in bacterial peptidoglycan biosynthesis and the source of activated N-acetylglucosamine in ... More
A phosphorylation-induced major structural change in the N-terminal domain of the P protein of Chandipura virus.
AuthorsRaha T, Chattopadhyay D, Chattopadhyay D, Roy S
JournalBiochemistry
PubMed ID10026294
'It has previously been shown that phosphorylation of P protein of vesicular stomatitis virus as well as Chandipura (CHP) virus is required for transcription activation and replication switch. The structural nature of this crucial conformational change, however, is largely unknown. We have studied the phosphorylation-associated conformational change in the P ... More
Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta.
AuthorsWolfgang CL, Zhang Z, Gabriel JL, Pieringer RA, Soprano KJ, Soprano DR
JournalJ Biol Chem
PubMed ID8995359
'The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss ... More
Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase.
AuthorsMas MT, Chen HH, Aisaka K, Lin LN, Brandts JF
JournalBiochemistry
PubMed ID7794905
'Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The ... More
Characterization of phospholipase A2 activity in reticulocyte endocytic vesicles.
AuthorsBette-Bobillo P, Vidal M
JournalEur J Biochem
PubMed ID7883004
'Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A2 activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spin-labeled phospholipid analogues, we measured the hydrolysis rate of the ester bond at position 2 during incubation with reticulocyte endocytic vesicles. We ... More
The reaction of superoxide with reduced glutathione.
AuthorsWinterbourn CC, Metodiewa D
JournalArch Biochem Biophys
PubMed ID7979367
'Superoxide, generated by a xanthine oxidase/hypoxanthine system, reacts with reduced glutathione (GSH) to cause an increase in oxygen consumption and oxidized glutathione (GSSG) formation, both of which are fully inhibited by superoxide dismutase. In this study we have shown that little, if any, of the additional oxygen consumed is converted ... More
Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase.
AuthorsEllis HR, Poole LB
JournalBiochemistry
PubMed ID9398227
'The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl ... More
Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina.
AuthorsWise ML, Rossi J, Gerwick WH
JournalBiochemistry
PubMed ID9062129
'The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be ... More
Fluorescent probes as a measure of conformational alterations induced by nucleophilic modification and proteolysis of bovine alpha 2-macroglobulin.
'Conformational alterations occurring in bovine alpha 2-macroglobulin (alpha 2M) resulting from proteolysis and nucleophilic modification have been monitored by UV difference spectra, circular dichroism, and changes in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and bis(8-anilino-1-naphthalenesulfonate) (Bis-ANS). The results of this study indicate that these two dyes appear capable of differentiating between ... More
Selective modulation of glutathione in mouse brain regions and its effect on acrylamide-induced neurotoxicity.
AuthorsShivakumar BR, Ravindranath V
JournalBiochem Pharmacol
PubMed ID1739414
'Selective modulation of brain glutathione (GSH) may assist the elucidation of the role of GSH in the central nervous system. Subcutaneous administration of diethyl maleate (DEM) depleted both cerebral and hepatic GSH in a dose- and time-dependent manner. While hepatic GSH levels returned to control levels 6 hr after DEM ... More
Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.
AuthorsMurphy LA, Goldstein IJ
JournalJ Biol Chem
PubMed ID68957
'The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated ... More
Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.
AuthorsSmith DJ, Maggio, ET, Kenyon GL
JournalBiochemistry
PubMed ID163643
'New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with ... More
Suppression of the antibody response to a polymorphic peptide from the platelet alloantigen integrin beta3 with low molecular weight antigen arrays.
AuthorsWatson DC, Reim J, Dintzis HM
JournalJ Immunol
PubMed ID8786303
'The allelic human platelet alloantigens PIA1/PIA2 are determined by a 33 Leu-Pro substitution in the second disulfide loop of the integrin beta3 subunit of the fibrinogen receptor, alphaIIb beta3 (GPIIb-IIIa). Alloantibodies to PIA1 cause neonatal alloimmune thrombocytopenia. We studied the suppression of specific Ab production to a disulfide-looped peptide spanning ... More
Location of conserved residue histidine-38 of the epsilon subunit of Escherichia coli ATP synthase.
AuthorsSkakoon EN, Dunn SD
JournalArch Biochem Biophys
PubMed ID7682392
'The function and location of residue His-38 of the epsilon subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce epsilon H38Q and epsilon H38C, respectively. Both epsilon H38Q and epsilon H38C fulfilled epsilon function in vivo as determined by ... More
Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer".
AuthorsHeintz D, Faulstich H
JournalBiochemistry
PubMed ID8555183
'Actin cross-linked between cys 374 and cys 10 via a disulfide-containing bridge, c-A, is completely unpolymerizable even in the presence of phalloidin. Upon the addition of dithiothreitol, c-A polymerizes with high yield, indicating that denaturation due to the modification was almost absent. In the present study we show that cross-linked ... More
S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain.
AuthorsTao L, Murphy ME, English AM
JournalBiochemistry
PubMed ID11994015
'Calbindin D(28K) is noted for its abundance and specific distribution in mammalian brain and sensory neurons. It can bind three to five Ca(2+) ions and may act as a Ca(2+) buffer to maintain intracellular Ca(2+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis ... More
Dual nanomolar and picomolar Zn(II) binding properties of metallothionein.
AuthorsKrezel A, Maret W,
JournalJ Am Chem Soc
PubMed ID17696343
'Each of the seven Zn(II) ions in the Zn(3)S(9) and Zn(4)S(11) clusters of human metallothionein is in a tetrathiolate coordination environment. Yet analysis of Zn(II) association with thionein, the apoprotein, and analysis of Zn(II) dissociation from metallothionein using the fluorescent chelating agents FluoZin-3 and RhodZin-3 reveal at least three classes ... More
Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex.
AuthorsPan CQ, Feng JA, Finkel SE, Landgraf R, Sigman D, Johnson RC
JournalProc Natl Acad Sci U S A
PubMed ID8127871
'The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates ... More
Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.
AuthorsKhan AR, Williams KA, Boggs JM, Deber CM
JournalBiochemistry
PubMed ID7547983
'The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein ... More
Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells.
AuthorsNair S, Singh SV, Krishan A
JournalCytometry
PubMed ID1648468
'We have used an enzymatic (spectro-photometric) and a flow cytometric (GSH-MBCL) method to compare the glutathione (GSH) content of doxorubicin sensitive (P388) and resistant (P388/R-84) murine leukemic and human lung cancer cells. The flow cytometric analysis revealed that GSH-MBCL conjugate formation was dependent on glutathione-S-transferase (GST) activity. The human solid ... More
Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase.
AuthorsYamazaki M, Ohnishi T, Ichikawa Y
JournalInt J Biochem
PubMed ID1733787
'1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5''-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5''-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of ... More
Nitric oxide donation and nitrite assays in the presence of thiols and albumin as determined by Griess' and Werringloer's methods.
AuthorsRobak J, Marcinkiewicz E, Michalska Z, Gryglewski RJ
JournalPol J Pharmacol
PubMed ID9437769
'Nitric oxide (NO) or nitrite (NO2-) were assayed using the Werringloer''s method or the Griess'' method, respectively, in the presence or absence of various thiols, amino acids, or albumin. This has been done because both methods are used to determine the generation of endogenous NO from L-arginine or exogenous NO ... More
Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.
AuthorsUbarretxena-Belandia I, Hozeman L, van der Brink-van der Laan E, Pap EH, Egmond MR, Verheij HM, Dekker N
JournalBiochemistry
PubMed ID10353852
'In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in ... More
A competitive fluorescence assay to measure the reactivity of compounds.
AuthorsEpps DE, Taylor BM
JournalAnal Biochem
PubMed ID11476550
'A sensitive competitive method was developed for assessing the reactivity of compounds toward glutathione and toward thiols in general. The method employs the reaction of the fluorogenic reagent fluorescein-5-maleimide (FM) with glutathione (GSH) to generate a large increase in fluorescence emission. When the reaction is measured in the presence of ... More
Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype.
AuthorsJazayeri M, Andreyev A, Will Y, Ward M, Anderson CM, Clevenger W,
JournalJ Biol Chem
PubMed ID12645575
'We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) ... More
Determination of reduced disulfide groups in monoclonal antibodies.
AuthorsThakur ML, DeFulvio JD
JournalBiotechniques
PubMed ID2357374
'Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which ... More
Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli.
AuthorsZantema A, Maassen JA, Kriek J, Möller W
JournalBiochemistry
PubMed ID6125208
'So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins. In the case of L7/L12, this was done by attaching an aldehyde ... More
The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding.
AuthorsBhattacharjee G, Asplin IR, Wu SM, Gawdi G, Pizzo SV
JournalJ Biol Chem
PubMed ID10862607
'alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these ... More
The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase.
AuthorsMittl PR, Lüthy L, Hunziker P, Grütter MG
JournalJ Biol Chem
PubMed ID10748053
'Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a ... More
Inhibition of the human sodium/bile acid cotransporters by side-specific methanethiosulfonate sulfhydryl reagents: substrate-controlled accessibility of site of inactivation.
AuthorsHallén S, Fryklund J, Sachs G
JournalBiochemistry
PubMed ID10828993
'Mammalian sodium/bile acid cotransporters (SBATs) constitute a subgroup of the sodium cotransporter superfamily and function in the enterohepatic circulation of bile acids. They are glycoproteins with an exoplasmic N-terminus, seven or nine transmembrane segments, and a cytoplasmic C-terminus. They exhibit no significant homology with other members of the sodium cotransporter ... More
A new and rapid colorimetric determination of acetylcholinesterase activity.
AuthorsEllman GL, Courtney KD, Andres V, Featherstone RM,
JournalBiochem Pharmacol
PubMed ID13726518
'Acetylthiocholine substrate cleavage assayed by spectrophotometric detection of thiocholine using DTNB.'
Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization.
Authorsvan Gijlswijk RP, van de Corput MP, Bezrookove V, Wiegant J, Tanke HJ, Raap AK
JournalHistochem Cell Biol
PubMed ID10817671
'A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for ... More
A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol.
AuthorsVandeputte C, Guizon I, Genestie-Denis I, Vannier B, Lorenzon G
JournalCell Biol Toxicol
PubMed ID7697505
'The microtiter plate technique reported by Baker and colleagues for the glutathione reductase-DTNB recycling assay of total glutathione (GSx) and glutathione disulfide (GSSG) has been modified according to Anderson''s recommendations, in order to improve the reliability and accuracy of this miniaturized method for the measurement of glutathione status in cultured/isolated ... More
Derivatization of thiol-containing compounds.
AuthorsShimada K, Mitamura K
JournalJ Chromatogr B Biomed Appl
PubMed ID7820279
'The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents ... More
Quantitation of hydrogen peroxide using tris(2-carboxyethyl)phosphine.
AuthorsHan J, Yen S, Han G, Han P
JournalAnal Biochem
PubMed ID8742092
Partial amino acid sequences around sulfhydryl groups of soybean beta-amylase.
AuthorsNomura K, Mikami B, Morita Y
JournalJ Biochem (Tokyo)
PubMed ID2444583
'Sulfhydryl (SH) groups of soybean beta-amylase were modified with 5-(iodoaceto-amidoethyl)aminonaphthalene-1-sulfonate (IAEDANS) and the SH-containing peptides exhibiting fluorescence were purified after chymotryptic digestion of the modified enzyme. The sequence analysis of the peptides derived from the modification of all SH groups in the denatured enzyme revealed the existence of six SH ... More
Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties.
AuthorsGuixé V
JournalArch Biochem Biophys
PubMed ID10775417
'Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of ... More
Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease.
AuthorsJha N, Jurma O, Lalli G, Liu Y, Pettus EH, Greenamyre JT, Liu RM, Forman HJ, Andersen JK
JournalJ Biol Chem
PubMed ID10846169
'Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson''s disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an ... More
Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism.
AuthorsZable AC, Favero TG, Abramson JJ
JournalJ Biol Chem
PubMed ID9054399
'In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on ... More
Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species.
'The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl). Protein conformations were characterized ... More
A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast.
AuthorsKampranis SC, Damianova R, Atallah M, Toby G, Kondi G, Tsichlis PN, Makris AM
JournalJ Biol Chem
PubMed ID10859306
'The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for ... More
Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.
AuthorsLing N, Shrimpton C, Sleep J, Kendrick-Jones J, Irving M
JournalBiophys J
PubMed ID8785344
'The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged ... More
Novel role for the NMDA receptor redox modulatory site in the pathophysiology of seizures.
AuthorsSanchez RM, Wang C, Gardner G, Orlando L, Tauck DL, Rosenberg PA, Aizenman E, Jensen FE
JournalJ Neurosci
PubMed ID10704515
'Redox-active compounds modulate NMDA receptors (NMDARs) such that reduction of NMDAR redox sites increases, and oxidation decreases, NMDAR-mediated activity. Because NMDARs contribute to the pathophysiology of seizures, redox-active compounds also may modulate seizure activity. We report that the oxidant 5, 5''-dithio-bis(2-nitrobenzoic acid) (DTNB) and the redox cofactor pyrroloquinoline quinone (PQQ) ... More
Thiol dependence of nitric oxide synthase.
AuthorsHofmann H, Schmidt HH
JournalBiochemistry
PubMed ID7577932
'Nitric oxide synthases (NOS) require NADPH and tetrahydrobiopterin (H4biopterin) to convert L-arginine to L-citrulline. The additional requirement and effects of thiols during purification and activity assays of NOS are unclear; for example, glutathione (GSH) has been reported to stimulate or, in the presence of catalase, to inhibit enzyme activity. We ... More
Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples.
AuthorsBaker MA, Cerniglia GJ, Zaman A
JournalAnal Biochem
PubMed ID2291479
'By combining the least complicated and expedient methods of sample handling with the sensitivity and specificity of the GSH assay by enzymatic recycling and the small volumes and software capabilities of microtiter plate technology we have devised a rapid, sensitive, and easy assay for GSH and GSSG in biological samples. ... More
Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion.
AuthorsMammoto A, Masumoto N, Tahara M, Yoneda M, Nishizaki T, Tasaka K, Miyake A
JournalJ Exp Zool
PubMed ID9181696
'Effects of various sulfhydryl (SH)-depleting reagents on sperm-egg fusion were demonstrated. When sperm were treated with three plasma membrane-permeable SH-depleting reagents, N-ethyl-maleimide, sodium tetrathionate and 5,5''-dithiobis (2-nitro-benzoic acid), the rates of cleavage of eggs were significantly lower than those with control sperm. Neither the motility, penetration of the zona pellucida, ... More
An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.
AuthorsHansen RE, Winther JR,
JournalAnal Biochem
PubMed ID19664585
'This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we ... More
Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values.
AuthorsShaked Z, Szajewski RP, Whitesides GM
JournalBiochemistry
PubMed ID6251863
'Brønsted coefficients have been determined for the rates of thiol-disulfide interchange between low molecular weight thiols and the disulfide groups of four native or modified proteins: DNase (beta nuc congruent to 0.36), lysozyme (beta nuc congruent to 0.55), adenylate kinase(SSCH3)2 (beta nuc congruent to 0.65), and papain(SSCH3) (beta nuc congruent ... More
Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1.
AuthorsSudharshan E, Rao AG
JournalJ Biol Chem
PubMed ID10585402
'Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the ... More
Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid.
AuthorsNishiyama J, Kuninori T
JournalAnal Biochem
PubMed ID6375458
'A simple and rapid method for the determination of nanomole levels of biological thiols is described. The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6''-dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting ... More
N-termini of EcoRI restriction endonuclease dimer are in close proximity on the protein surface.
'The N-terminal region of EcoRI endonuclease is essential for cleavage yet is invisible in the 2.5 A crystal structure of endonuclease-DNA complex [Kim, Y., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990) Science 249, 1307-1309]. We used site-directed fluorescence spectroscopy and chemical cross-linking to locate the ... More
Spectrophotometric assay of thiols.
AuthorsJocelyn PC
JournalMethods Enzymol
PubMed ID3657559
Measurement of protein thiol groups and glutathione in plasma.
AuthorsHu ML
JournalMethods Enzymol
PubMed ID8015473
Determination of biothiols by bromobimane labeling and high-performance liquid chromatography.
AuthorsNewton GL, Fahey RC
JournalMethods Enzymol
PubMed ID7651194
Glutathione and cellular resistance to anti-cancer drugs.
Peroxynitrite was demonstrated to inhibit the active efflux of glutathione S-conjugates (2,4-dinitrophenyl-S-glutathione and bimane-S-glutathione) from human erythrocytes and the erythrocyte membrane ATPase activity stimulated by glutathione S-conjugates. As the multidrug resistance-associated protein (MRP) is responsible for the transport of glutathione S-conjugates in mammalian cells, these results point to the possibility ... More
Quantitative determination of thiolipids in organic solution, in membranes, and on HPTLC plates.
AuthorsZellmer S, Lasch J, Rothe U
JournalJ Lipid Res
PubMed ID9392435
In the presence of ortho-phthalaldehyde and glucosamine, thiolipids form fluorescent isoindole derivatives. This reaction can be used to quantify single- and double-chain mercaptans in membranes (liposomes) and micellar solutions. The lower detection limit is 100 pmol. In addition, the assay allows the detection of 1.9 nmol thiolipids on HPTLC plates ... More
S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control.
A dynamic cycle exists in which haemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated, and the NO group is released during arterial-venous transit. The vasoactivity of S-nitrosohaemoglobin is promoted by the erythrocytic export of S-nitrosothiols. These findings highlight newly discovered allosteric and electronic properties of haemoglobin ... More
Sulfhydryl groups are involved in H+ translocation via the uncoupling protein of brown adipose tissue mitochondria.
AuthorsJezek P
JournalFEBS Lett
PubMed ID3026844
Mersalyl inhibits H+ transport via the uncoupling protein (UP) in brown adipose tissue (BAT) mitochondria estimated as swelling in potassium acetate (Ki 67 microM) or as valinomycin-induced H+ extrusion in K2SO4 (Ki 55 microM) and KCl. The swelling in KCl is depressed only slightly. Some other SH-reagents (p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate) and ... More
A mutant truncated protein disulfide isomerase with no chaperone activity.
AuthorsDai Y, Wang C
JournalJ Biol Chem
PubMed ID9346892
A mutant human protein disulfide isomerase with the COOH-terminal 51 amino acid residues deleted (abb'a') has been expressed in Escherichia coli. Its secondary structures are very similar to those of the native bovine enzyme. The mutant enzyme shows neither peptide binding ability nor chaperone activity in assisting the refolding of ... More
Synthesis and applications of a new poly(ethylene glycol) derivative for the crosslinking of amines with thiols.
AuthorsHaselgrübler T, Amerstorfer A, Schindler H, Gruber HJ
JournalBioconjug Chem
PubMed ID7632794
A heterobifunctional crosslinker was synthesized from a diamine derivative of poly(ethylene glycol) (PEG, average molecular weight 800 Da), with the functional groups 2-(pyridyldithio)propionyl (PDP) and N-hydroxysuccinimide ester (NHS). The crosslinker can be used for linkage of two different proteins for which a suitable protocol is presented, explified by crosslinking of ... More
Features of a spatially constrained cystine loop in the p10 FAST protein ectodomain define a new class of viral fusion peptides.
AuthorsBarry C, Key T, Haddad R, Duncan R,
JournalJ Biol Chem
PubMed ID20363742
The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue ... More
Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.
AuthorsHuang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall JD, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AI
JournalJ Biol Chem
PubMed ID10601271
Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by ... More
Cysteinyl residues of Escherichia coli recA protein.
The Escherichia coli recA protein has three cysteinyl residues at positions 90, 116, and 129. All of them are reactive with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). In the presence of ATP or ADP, only one cysteinyl residue reacts with DTNB. The residue was also reactive with N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide (DACM) in the presence of ... More
Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled ... More
Two-dimensional arrangement of a functional protein by cysteine-gold interaction: enzyme activity and characterization of a protein monolayer on a gold substrate.
AuthorsSasaki YC, Yasuda K, Suzuki Y, Ishibashi T, Satoh I, Fujiki Y, Ishiwata S
JournalBiophys J
PubMed ID9083688
We have characterized the functional protein, myosin subfragment 1 (S1), attached to a gold substrate by the sulfhydryl groups of cysteine in proteins. The amino groups of the regulatory light chain (RLC) isolated from myosin were labeled with a radioisotope (125I), and the labeled RLC was incorporated into S1 from ... More
Studies on the identity of the heme-binding cysteinyl residue in rabbit liver microsomal cytochrome P-450 isozyme 2.
AuthorsBlack SD, Coon MJ
JournalBiochem Biophys Res Commun
PubMed ID3985983
The reaction of purified rabbit liver microsomal P-450 isozyme 2 with 4,4'-dithiobis(2-nitrobenzoate) (DTNB) exhibits first order kinetics and results in the modification of a single thiol, but causes no net loss of the native ferrous-carbonyl spectrum. Inclusion of both phospholipid and a tight-binding nitrogenous ligand, 1-benzylimidazole, in the reaction medium ... More
Glutathione suppresses spontaneous activity in the frog spinal cord.
AuthorsLoguercio C, Di Cosmo A, De Santis A, Nardi G
JournalNeuroreport
PubMed ID8527738
Spontaneous electrical activity in the isolated hemisected frog spinal cord increased in the presence of the SH reductant dithiothreitol (DTT) was reversibly suppressed by the oxidant 5-5'-dithio-bis-(2 nitrobenzoic acid) (DTNB) and was irreversibly suppressed by the sulphydryl modifying agents N-ethyl-maleimide (NEM) and monobromotrimethylammonio bimane. Glutathione (GSH), an important natural low ... More
Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.
We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the ... More
Suppression of microtubule dynamics by LY290181. A potential mechanism for its antiproliferative action.
AuthorsPanda D, Singh JP, Wilson L
JournalJ Biol Chem
PubMed ID9065425
LY290181, 2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile, is a potent antiproliferative compound that blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. In the present work we found that LY290181 bound to tubulin with high affinity (1 mol of LY290181 per mol of tubulin dimer; Ka, 3.8 x ... More
Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
AuthorsCardemil E, Encinas MV, Jabalquinto AM
JournalBiochim Biophys Acta
PubMed ID2198945
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), ... More
A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
AuthorsWu J, Gage DA, Watson JT
JournalAnal Biochem
PubMed ID8833324
A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) ... More
Fluorescence probes in biochemistry: an examination of the non-fluorescent behavior of dansylamide by photoacoustic calorimetry.
AuthorsGrabowski JJ, Bertozzi CR, Jacobsen JR, Jain A, Marzluff EM, Suh AY
JournalAnal Biochem
PubMed ID1481973
Photoacoustic calorimetry is shown to be a simple, precise, and accurate method for the quantification of the photophysics of a fluorescence probe, e.g., dansylamide, in a variety of solvents. This technique, which is described in detail, provides a direct measurement of the energy that is released nonradiatively following photostimulation, and ... More
L-Carnitine L-tartrate supplementation favorably affects markers of recovery from exercise stress.
We examined the influence of L-carnitine L-tartrate (LCLT) on markers of purine catabolism, free radical formation, and muscle tissue disruption after squat exercise. With the use of a balanced, crossover design (1 wk washout), 10 resistance-trained men consumed a placebo or LCLT supplement (2 g L-carnitine/day) for 3 wk before ... More
Sulfhydryl modification of E. coli Cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex.
AuthorsMendoza JA, Horowitz PM
JournalJ Protein Chem
PubMed ID1361328
Differential chemical modification of E. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were ... More
Analytical methods to investigate glutathione and related compounds in biological and pathological processes.
AuthorsCamera E, Picardo M
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12450659
Reduced glutathione (GSH, gamma-L-glutamyl-L-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in ... More
Optimized procedures for the coupling of proteins to liposomes.
AuthorsLoughrey HC, Choi LS, Cullis PR, Bally MB
JournalJ Immunol Methods
PubMed ID2391438
A general, optimized method for coupling proteins to liposomes is presented. This procedure utilizes streptavidin covalently coupled to liposomes to allow the subsequent attachment of a variety of biotinated proteins of interest. In the first part of this study, covalent methods for coupling proteins to liposomes which contain the lipid ... More
Sensitive and selective flow injection analysis of hydrogen sulfite/sulfur dioxide by fluorescence detection with and without membrane separation by gas diffusion.
AuthorsMana H, Spohn U
JournalAnal Chem
PubMed ID11467572
Highly sensitive and selective FIA flow injection analysis procedures for the determination of sulfite/hydrogen sulfite/sulfur dioxide were developed on the basis of an in situ-generated o-phthalaldehyde (OPA)/ammonium reagent and fluorescence detection. The highest sensitivity was achieved at an excitation wavelength of 330 nm, an emission wavelength of 390 nm, and ... More
N-acetylcysteine-promoted survival of PC12 cells is glutathione-independent but transcription-dependent.
AuthorsYan CY, Ferrari G, Greene LA
JournalJ Biol Chem
PubMed ID7592924
Our prior work established that comparable concentrations of N-acetylcysteine (NAC) both block the proliferation of PC12 cells and prevent death of trophic factor-deprived sympathetic neurons and PC12 cells. The present work addresses several aspects of the mechanisms of these actions. NAC increases intracellular levels of glutathione (GSH) by approximately 10-fold ... More
Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.
AuthorsRhee SS, Burke DH
JournalAnal Biochem
PubMed ID14715294
We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] ... More
Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.
AuthorsRadi R, Beckman JS, Bush KM, Freeman BA
JournalJ Biol Chem
PubMed ID1847917
Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for ... More
Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells.
The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair ... More