Free radical-induced inactivation of creatine kinase: influence on the octameric and dimeric states of the mitochondrial enzyme (Mib-CK)

Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D37-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mib-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mib-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.

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Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

Biochemical Journal ◽

10.1042/bj2910103 ◽

1993 ◽

Vol 291 (1) ◽

pp. 103-107 ◽

Cited By ~ 64

Author(s):

H M Zhou ◽

X H Zhang ◽

Y Yin ◽

C L Tsou

Keyword(s):

Creatine Kinase ◽

Fluorescent Probe ◽

Conformational Change ◽

Conformational Changes ◽

Active Site ◽

Emission Intensity ◽

Enzyme Inactivation ◽

Amino Groups ◽

Guanidinium Chloride ◽

Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

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Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue

Biochemical Journal ◽

10.1042/bj3470821 ◽

2000 ◽

Vol 347 (3) ◽

pp. 821-827 ◽

Cited By ~ 44

Author(s):

Sharanya REDDY ◽

A. Daniel JONES ◽

Carroll E. CROSS ◽

Patrick S.-Y. WONG ◽

Albert VAN DER VLIET

Keyword(s):

Oxidative Stress ◽

Creatine Kinase ◽

Molecular Mass ◽

Active Site ◽

Protein S ◽

Cysteine Residue ◽

Enzyme Inactivation ◽

Ischaemic Injury ◽

Active Site Cysteine ◽

Active Site Cysteine Residue

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys283) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.

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Electron Paramagnetic Resonance Spectrometry-based Assessment of Free Radicals Scavenging Potential of N-acetyl Tryptophan Glucoside

Defence Life Science Journal ◽

10.14429/dlsj.2.11672 ◽

2017 ◽

Vol 2 (3) ◽

pp. 317 ◽

Cited By ~ 1

Author(s):

Poonam Malhotra ◽

Yana Karamalakova ◽

Galina Nikolova ◽

Darshana Singh ◽

Raj Kumar

Keyword(s):

Free Radicals ◽

Free Radical ◽

Gamma Radiation ◽

Radical Scavenging ◽

Control Group ◽

Water Radiolysis ◽

In Vivo Models ◽

Radiation Induced ◽

Free Radicals Scavenging

<p>Gamma radiation generates free radicals in biological system by inducing cellular water radiolysis. If not neutralised, free radicals oxidise vital bio-macromolecules causing structural and functional impairment and contribute to cell death. In present study, free radical scavenging activities of a novel bacterial secondary metabolite, N-acetyl tryptophan glucoside (NATG) was assessed against gamma-radiation induced damage in vitro and in vivo models. Effect of irradiated NATG (UV and gamma radiation 8 Gy and 20 Gy) on its free radical (DPPH radicals) and SOD-like activity was evaluated using EPR spectrometry. To assess the effect of NATG irradiation on its antioxidant potential, EPR based ascorbate, PBN and NO radicals scavenging activities were evaluated in blood and spleen tissue of strain A male mice. Results of the study indicated significant (p < 0.05) increase in DPPH radicals scavenging ability of irradiated NATG as compared to un-irradiated NATG. Similarly, irradiated NATG exhibited significant (p < 0.05) elevation in SOD-like activity as compared to control. Subsequently, NATG treatment displayed enhanced antioxidant activity as evident by significant (p < 0.05) decline in ascorbate, PBN and NO radicals at 1 h and 2h in blood and spleen tissues homogenate of treated mice as compared to control group. In conclusion, NATG possesses significant free radicals scavenging and radio protective capabilities against gamma radiation induced oxidative stress.</p>

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Monte Carlo simulation study of the effects of acidity and LET on the primary free-radical and molecular yields of water radiolysis — Application to the Fricke dosimeter

Canadian Journal of Chemistry ◽

10.1139/v07-021 ◽

2007 ◽

Vol 85 (3) ◽

pp. 214-229 ◽

Cited By ~ 22

Author(s):

Narongchai Autsavapromporn ◽

Jintana Meesungnoen ◽

Ianik Plante ◽

Jean-Paul Jay-Gerin

Keyword(s):

Monte Carlo ◽

Energy Transfer ◽

Free Radicals ◽

Sulfuric Acid ◽

Free Radical ◽

Monte Carlo Simulations ◽

Linear Energy Transfer ◽

Oh Radicals ◽

Water Radiolysis ◽

Monte Carlo Simulation Study

Monte Carlo simulations are used to investigate the effects of acidity (pH) on the primary yields of various chemical species produced in the radiolysis of de-aerated aqueous sulfuric acid solutions over the range from neutral solution to 0.4 mol/L H2SO4. The effects of the quality of radiation, measured in terms of linear energy transfer (LET), have also been studied for LET varying from ~0.3 to 15 keV/µm at ambient temperature. Our results show that an increase in acidity (1 < pH < 4) leads to an increase in the yield [Formula: see text] of the "reducing" free radicals (hydrated electron and H atom) and a slight increase in G·OH and [Formula: see text], while there is a slight decrease in [Formula: see text] At pH < 1, OH radicals react with HSO4- anions to form SO4·– radicals, resulting in a steep decrease in G.OH. By contrast, in the range of pH from ~4 to 7, the calculated yield values are independent of sulfuric acid concentration. In both neutral water and 0.4 mol/L H2SO4 (pH 0.46) solutions, the primary molecular yields increase upon increasing LET to ~15 keV/µm with a concomitant decrease in those of free radicals. As an exception, GH. at first increases with LET, reaching a maximum near 6.5 keV/µm before decreasing steeply at higher LET. The results obtained are generally in good agreement with available experimental data over the whole acidity and LET ranges studied. Finally, as an application, we have simulated the radiation-induced oxidation of ferrous sulfate solutions in aerated aq. 0.4 mol/L H2SO4 (Fricke dosimeter) as a function of time up to ~50 s and addressed the effects of LET on the resulting ferric ion yield at 25 °C. The production of Fe3+ ions is highly sensitive to free-radical yields, especially H atoms (via formation of HO2), resulting in a marked decline of G(Fe3+) with increasing LET. The general trend of the observed variation of G(Fe3+) with radiation quality is well reproduced by our computed Fe3+ ion yield values.Key words: liquid water, acidic (H2SO4) aqueous solutions, radiolysis, free-radical and molecular yields, linear energy transfer (LET), Fricke dosimeter, Monte Carlo simulations.

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Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction

10.1101/472381 ◽

2018 ◽

Author(s):

Sébastien Triboulet ◽

Zainab Edoo ◽

Fabrice Compain ◽

Clément Ourghanlian ◽

Adrian Dupuis ◽

...

Keyword(s):

Fluorescence Quenching ◽

Active Site ◽

Tryptophan Fluorescence ◽

Multidrug Resistant ◽

Enzyme Inactivation ◽

Cross Linking ◽

Mesomeric Effect ◽

Resistant Tuberculosis ◽

The Status ◽

Covalent Adducts

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

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Study of antioxidant potential in leaves, stems, nuts of Juglans regia L.

International Journal of Bioassays ◽

10.21746/ijbio.2012.10.003 ◽

2012 ◽

Vol 1 (10) ◽

pp. 79 ◽

Cited By ~ 2

Author(s):

G. Raja* ◽

Ivvala Anand Shaker ◽

Inampudi Sailaja ◽

R. Swaminathan ◽

S. Saleem Basha ◽

...

Keyword(s):

Free Radicals ◽

Free Radical ◽

Antioxidant Activities ◽

Radical Scavenging Activity ◽

Juglans Regia ◽

Radical Scavenging ◽

Antioxidant Compounds ◽

Free Radical Damage ◽

Radical Damage

Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods. The role of antioxidants has protected effect against free radical damage that may cause many diseases including cancer. Primary sources of naturally occurring antioxidants are known as whole grains, fruits, and vegetables. Several studies suggest that regular consumption of nuts, mostly walnuts, may have beneficial effects against oxidative stress mediated diseases such as cardiovascular disease and cancer. The role of antioxidants has attracted much interest with respect to their protective effect against free radical damage that may cause many diseases including cancer. Juglans regia L. (walnut) contains antioxidant compounds, which are thought to contribute to their biological properties. Polyphenols, flavonoids and flavonols concentrations and antioxidant activity of Leaves, Stems and Nuts extract of Juglans regia L. as evaluated using DPPH, ABTS, Nitric acid, hydroxyl and superoxide radical scavenging activity, lipid peroxidation and total oxidation activity were determined. The antioxidant activities of Leaves, Stems and Nuts extract of Juglans regia L. were concentration dependent in different experimental models and it was observed that free radicals were scavenged by the test compounds in all the models.

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THE KINETICS OF THE THERMAL REACTIONS OF ETHYLENE OXIDE

Canadian Journal of Chemistry ◽

10.1139/v63-434 ◽

1963 ◽

Vol 41 (12) ◽

pp. 2956-2961 ◽

Cited By ~ 16

Author(s):

M. Lynne Neufeld ◽

Arthur T. Blades

Keyword(s):

Free Radicals ◽

Free Radical ◽

Ethylene Oxide ◽

Thermal Reactions ◽

Kinetics Of

The thermal reactions of ethylene oxide in the presence of an excess of propylene have been studied as a function of pressure and it has been found that there are two sets of products, acetaldehyde and free radicals, presumably methyl and formyl. These products are believed to arise from an excited acetaldehyde intermediate. Some evidence has been obtained for the occurrence of a surface-catalyzed rearrangement to acetaldehyde but the free radical products are uninfluenced by surface.

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Repeated Administration of Clinically Relevant Doses of the Prescription Opioids Tramadol and Tapentadol Causes Lung, Cardiac, and Brain Toxicity in Wistar Rats

Pharmaceuticals ◽

10.3390/ph14020097 ◽

2021 ◽

Vol 14 (2) ◽

pp. 97

Author(s):

Joana Barbosa ◽

Juliana Faria ◽

Fernanda Garcez ◽

Sandra Leal ◽

Luís Pedro Afonso ◽

...

Keyword(s):

Creatine Kinase ◽

Wistar Rats ◽

Brain Cortex ◽

Dose Range ◽

Fibrous Tissue ◽

Lactate Concentration ◽

Repeated Administration ◽

Serum Lactate Dehydrogenase ◽

Prescription Opioids ◽

Therapeutic Doses

Tramadol and tapentadol, two structurally related synthetic opioid analgesics, are widely prescribed due to the enhanced therapeutic profiles resulting from the synergistic combination between μ-opioid receptor (MOR) activation and monoamine reuptake inhibition. However, the number of adverse reactions has been growing along with their increasing use and misuse. The potential toxicological mechanisms for these drugs are not completely understood, especially for tapentadol, owing to its shorter market history. Therefore, in the present study, we aimed to comparatively assess the putative lung, cardiac, and brain cortex toxicological damage elicited by the repeated exposure to therapeutic doses of both prescription opioids. To this purpose, male Wistar rats were intraperitoneally injected with single daily doses of 10, 25, and 50 mg/kg tramadol or tapentadol, corresponding to a standard analgesic dose, an intermediate dose, and the maximum recommended daily dose, respectively, for 14 consecutive days. Such treatment was found to lead mainly to lipid peroxidation and inflammation in lung and brain cortex tissues, as shown through augmented thiobarbituric acid reactive substances (TBARS), as well as to increased serum inflammation biomarkers, such as C reactive protein (CRP) and tumor necrosis factor-α (TNF-α). Cardiomyocyte integrity was also shown to be affected, since both opioids incremented serum lactate dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (α-HBDH) activities, while tapentadol was associated with increased serum creatine kinase muscle brain (CK-MB) isoform activity. In turn, the analysis of metabolic parameters in brain cortex tissue revealed increased lactate concentration upon exposure to both drugs, as well as augmented LDH and creatine kinase (CK) activities following tapentadol treatment. In addition, pneumo- and cardiotoxicity biomarkers were quantified at the gene level, while neurotoxicity biomarkers were quantified both at the gene and protein levels; changes in their expression correlate with the oxidative stress, inflammatory, metabolic, and histopathological changes that were detected. Hematoxylin and eosin (H & E) staining revealed several histopathological alterations, including alveolar collapse and destruction in lung sections, inflammatory infiltrates, altered cardiomyocytes and loss of striation in heart sections, degenerated neurons, and accumulation of glial and microglial cells in brain cortex sections. In turn, Masson’s trichrome staining confirmed fibrous tissue deposition in cardiac tissue. Taken as a whole, these results show that the repeated administration of both prescription opioids extends the dose range for which toxicological injury is observed to lower therapeutic doses. They also reinforce previous assumptions that tramadol and tapentadol are not devoid of toxicological risk even at clinical doses.

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