The oxidation of aminoethylcysteine ketimine dimer by oxygen reactive species

Summary The prominent spontaneous reaction of aminoethylcysteine ketimine in the neutral pH range is the concentration-dependent dimerization (Hermann, 1961). The carboxylated dimer first produced loses the free carboxyl yielding the more stable decarboxylated dimer (named simply the dimer in this note). In the search for a possible biochemical activity of this uncommon tricyclic compound we have assayed whether it could interact with oxygen reactive species (H₂O₂, O₂ ^–,OH) thus exhibiting a scavenging effect of possible biomedical interest. The dimer interacts with H₂O₂ producing compounds detectable by chromatographic procedures. The presence of Fe²⁺ stimulates the oxidative reaction by yielding the hydroxyl radical (the Fenton reaction). Using the system xanthine oxidase-xanthine as superoxide producer, the dimer oxidation by O₂ ^– has also been documented. Among the oxidation products the presence of taurine and cysteic acid has been established. Identification of remaining oxidation products and investigation of the possible function of the dimer as a biological scavenger of oxygen reactive species are now oncoming.Abbreviations HPLC high performance liquid chromatography - AAÅ amino acid analyzer - SOD superoxide dismutase - EDTA ethylenediaminetetraacetic acid     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.     

Functional sulfhydryl groups of ferredoxin-NADP+ oxidoreductase

Chemical modification of membrane-bound ferredoxin-NADP⁺ oxidoreductase with oxidants of vicinal dithiols caused inactivation of NADP⁺ photoreduction, with no effect on the diaphorase activity. Inactivation was partially prevented by ferredoxin and reversed by dithioerythritol. N-Ethylmaleimide inhibited both activities, even though with a different kinetic pattern. Inactivation of NADP⁺ reduction by either N-ethylmaleimide or o-iodosobenzoate was greater in the light than in the dark. The results suggest the existence of essential sulfhydryl groups related with the ferredoxin site, in addition to those described in the soluble flavorprotein. The role of SH residues in the activity and regulation of membrane bound reductase is discussed.     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H⁺). Although its acid-base properties were indistinguishable from those of δ-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the δ-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0.It is apparent that the assay of δ-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. IV. Effect of surface-active agents on the antimycin-inhibition curve and availability of sulfhydryl groups of the heart muscle preparation

1. Pretreatment of submitochondrial particles with anionic detergents, such as deoxycholate and dodecyl sulfate, results in a change in the curve describing inhibition by antimycin of the succinate-cytochrome c reductase from sigmoidal towards linear.

2. On treatment of the preparation with either nonionic (Triton X-100 or Tween 80) or cationic (Cetavlon) detergents, the sigmoidal inhibition curve is retained. However, the preparation preincubated with Tween 80 is one half as sensitive to antimycin as the untreated one despite the fact that the activity of the preparation is not affected by this detergent.

3. In the presence of the anionic detergents, much higher amounts of sulfhydryl groups of the preparation are titratable by 5,5′-dithiobis(2-nitrobenzoic acid) than those of the control preparation. Addition of antimycin is without effect.

4. Preincubation of the preparation with Cetavlon results in only a small increase in the amount of sulfhydryl groups, whereas the nonionic detergents are without effect on the sulfhydryl content of the preparation.

5. The results indicate that the anionic detergents at the concentration transforming the antimycin-inhibition curve from sigmoidal towards linear result in a rapid increase of the sulfhydryl content of the heart-muscle preparation.     

The sulfhydryl groups of the thiol-dependent cytolytic toxin from Bacillus alvei evidence for one essential sulfhydryl group

Alveolysin, an extracellular protein toxin (M_r ? 63,000) excreted by Bacillus alvei and purified to homogeneity was shown to contain four cysteine residues. All thiol groups of the hemolytically active toxin preparation were free as found by direct titration by 5,5′-dithiobis (2-nitrobenzoic acid) and confirmed by the absence of disulfide bond. Toxin alkylation with tosyl lysine chloromethyl ketone resulted in the complete loss of hemolytic activity and the disappearance of only one thiol group with no modification of histidine residues. These results support the conclusion that one essential thiol group is implicated in the membrane-disrupting activity of alveolysin.     

Glutaredoxin 2 prevents H2O2-induced cell apoptosis by protecting complex I activity in the mitochondria

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H₂O₂) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H₂O₂-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H₂O₂ treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H₂O₂-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H₂O₂-induced apoptosis is likely associated with its ability to preserve complex I.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Site of red cell cation leak induced by mercurial sulfhydryl reagents

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS ($\text{p-}\text{chloromercuribenzene sulfonate}$) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that $\text{N-}\text{ethylmaleimide}$, a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.     

A subclass of albumin recognized by inhibition of phosphatidylethanolamine-mediated agglutination of mouse erythrocytes

Agglutination of mouse erythrocytes by non-choline phospholipids is inhibited by a factor in mammalian sera. The inhibitor cochromatographed with albumin on dye-agarose conjugates, was retained by an anti-albumin affinity column, was neutralized by anti-albumin antibody and found in a serum fraction in which only albumin could be detected. A variety of commercial preparations of albumin (fraction V, crystalline) did not inhibit. However, they acquired potent inhibitory activity when treated with low molecular weight thiols. The inhibitory activity of serum was increased 8-fold by treatment with dithiothreitol. Other proteins were not activated in this way. Inhibitory activity increased with average free sulphydryl content of treated albumin, up to six thiol groups per molecule. Alkylation of these sulphydryl groups did not diminish inhibitory activity. Thiols also induced polymerization of albumin. Inhibitory albumin in serum was largely monomeric. We propose that the inhibitor is a type of serum albumin which is lost or inactivated during preparation of commercial albumin, and which shares a structural feature, necessary for inhibition, with thiol-reduced albumin and the ligand on mouse erythrocytes.     

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

Molecular and structural basis for redox regulation of beta-actin

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H₂O₂. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H₂O₂ acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca²⁺. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H₂O₂ in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H₂O₂. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Chlorophyll destruction in the presence of bisulfite and linoleic acid hydroperoxide

Chlorophyll was rapidly destroyed in the presence of bisulfite and linoleic acid hydroperoxide. Both bisulfite and linoleic acid hydroperoxide were required for chlorophyll destruction and both were consumed in the reaction; however, there was no oxygen requirement. Chlorophyll destruction occurred most readily in the slightly acidic pH region with little destruction occurring above pH 8. The free radical scavengers, hydroquinone and α-tocopherol, were very effective at inhibiting chlorophyll destruction, but the singlet oxygen quenchers, β-carotene, 2,5-dimethylfuran and 1,3-diphenylisobenzofuran, were only slightly effective. The addition of metal chelators indicated that metals were not participating in the reaction. The evidence indicates that chlorophyll was destroyed by a free radical mechanism. Based on the present results and that of others, it is suggested that chlorophyll was destroyed via oxidation by the alkoxy radical which was produced during the decomposition of linoleic acid hydroperoxide by bisulfite.     

Structural requirements of alkyl acyldithiocarbazates for the uncoupling of oxidative phosphorylation in mitochondria

A structure-activity relationship study on the uncoupling of alkyl acyldithiocarbazates was carried out. Greater activity was observed with increasing alkyl chain length, the optimum being C₉. A further increase in alkyl chain length caused a decrease in the activity. Thione-thiol tautomeric forms with a dissociable proton were found to be of primary importance for the uncoupling and the role of the acyl group was auxiliary.The reactivity of the SH group of alkyl acyldithiocarbazates with an SH-reagent was very low. These compounds facilitated the valinomycin-induced swelling of non-respiring mitochondria and non-sonicated lecithin liposomes in isotonic potassium acetate solution.