[Show abstract][Hide abstract] ABSTRACT: The effect of polyhydroxylated fullerene (fullerenol), C60(OH)36, on human peripheral blood mononuclear cells (PBMCs) exposed to X-rays was studied. PBMCs untreated and treated for 1 h with C60(OH)36 at the concentrations 75 and 150 mg/l were exposed to high doses of ionizing radiation (10, 30 and 50 Gy). After 24 and 48 h of post-irradiation incubation the viability and granularity of lymphocytes were determined applying the flow cytometry (FC) method. Moreover, after 24 h of incubation the membrane fluidity was investigated by measuring the fluorescence anisotropy of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. Additionally, DNA damage of PBMCs after exposure to X-rays at the doses 0, 5, 10 and 15 Gy in the absence and presence of fullerenol (75 mg/l) was determined using the comet assay under alkaline conditions. Results show that the effects of fullerenol C60(OH)36 on X-irradiated human PBMCs are very small or inexistent. It was suggested that this action of C60(OH)36 may be related to its interactions with the surface of plasma membrane but not inside PBMCs.
Radiation Physics and Chemistry 04/2014; 97:325-331. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study the effect of X-irradiation on the alcohol dehydrogenase (ADH) activity in the presence of nanoparticles of fullerenol C60(OH)~30 under aerobic conditions was investigated in order to assess the potential radioprotective properties of fullerenol.
Fullerenol at 75 mg/mL decreased the radiation yield of inactivation of ADH irradiated with fullerenol by 20% comparing to ADH irradiated without fullerenol. Under conditions used during irradiation, 50% of radical dotOH radicals could react with fullerenol and 50% could react with ADH. Thus, it can be assumed that protective effect of fullerenol on the radiation inactivation of ADH was mostly due to scavenging radical dotOH radicals by fullerenol. Moreover, fullerenol did not protect against post-irradiation damage as the Ginact for ADH irradiated with fullerenol was still 20% lower than for ADH irradiated without fullerenol after 24 h from irradiation. Additionally, fullerenol at 75 mg/L had no influence on the activity of unirradiated ADH up to 24 h.
We concluded that fullerenol C60(OH)~30 protected ADH against radiation inactivation due to simple competition for the radical dotOH radicals and did not modify its activity by association with the protein as it was proved in our previous papers for erythrocyte membrane proteins.
Radiation Physics and Chemistry 04/2014; 97:102–106. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key redox-sensitive protein, the activity of which is largely affected by oxidative modifications at its highly reactive cysteine residue in the active site of the enzyme (Cys-152). These modifications occur as a result of S-thiolation, S-nitrosylation or disulfide bonds that lead to aggregate formation. The oxidative changes not only affect the glycolytic function but also stimulate the participation of GAPDH in numerous cellular processes. In this review we describe how thiol modification of Cys-152 in GAPDH re-routes metabolic pathways in the cell and converts a metabolic enzyme into a pro-apoptotic factor. Especially interesting issue is the participation of GAPDH in the regulation of expression of endothelin 1 and nitrosylation of nuclear proteins. In the last section we describe involvement of GAPDH in the processes associated with neurodegenerative diseases.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 01/2014; 68:280-90.
[Show abstract][Hide abstract] ABSTRACT: Protein damage mediated by oxidation has been associated with aging and age-related diseases, in particular neurodegenerative diseases. The protein that is known to be one of the major targets of oxidative stress is glyceraldehyde-3-phosphate dehydrogenase. GAPDH is believed to play a key role in certain neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Several recent studies have suggested that a wide range of variety of polyphenols including resveratrol may have neuroprotective effects. Here, we present data that clearly indicate the prooxidative properties of resveratrol and tiron in the inactivation of GAPDH induced by the superoxide anion generated via xanthine oxidase mediated oxidation of xanthine. Generated in the studied system tiron and resveratrol radicals are much more efficient in the inactivation of GAPDH than the superoxide anion alone. The analysis of CD spectra of protein exposed to the tiron and resveratrol radicals revealed little effect on the secondary structure of GAPDH. In both cases reduction of α-helical structure was followed by the increase in β-sheet conformation. Thus, the most probable mechanism of inactivation of GAPDH in the studied system is oxidation of cysteine residues in the catalytic center of the enzyme. Finally, molecular modeling of the resveratrol - GAPDH and tiron - GAPDH complexes showed potential binding sites for those antioxidants with binding affinity -45 kcal/mol and -48 kcal/mol respectively.
Current Medicinal Chemistry 09/2013; · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Purpose: Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. Material/Methods: Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO2, GSH, fluorescent label DCFH2) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. Results: The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO2 and DCFH2 was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h-1) was 50% lower than that of the control while the HbO2 oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. Conclusions: Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO2 and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.
Advances in Medical Sciences 05/2013; 58(1):134-142. · 0.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For a long time glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein of little interest. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. However, recent evidence demonstrates that GAPDH from mammalian cells displays a number of diverse activities unrelated to its glycolytic function. This enzyme is an example of moonlighting protein. Dehydrogenase participates in membrane fusion, microtubule assembly, vesicular transport, and the maintenance of DNA integrity. New and novel studies indicate that enzyme is directly involved in transcriptional, posttranscriptional gene regulation, and the maintenance of chromatin structure. Furthermore, other studies also indicate a role of GAPDH in apoptosis, and age-related neurodegenerative disease e.g. Alzheimer's, Huntington's and Parkinson's diseases. This work describes the structure and localization of GAPDH in cells as well as the latest discoveries on the multifunctional properties of the enzyme.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 01/2013; 67:775-89.
[Show abstract][Hide abstract] ABSTRACT: Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure.
Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (–SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.
Radiation Physics and Chemistry 04/2012; 81(7):807-815. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as OH and ONOO‐.In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH).We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion.Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.
Radiation Physics and Chemistry 01/2010; · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: W niniejszej pracy oceniano uszkodzenia erytrocytów człowieka indukowane w obecności mannitolu lub etanolu przez radiacyjnie generowane RFT oraz podczas przechowywania (głodzenia) erytrocytów. Napromieniowane promieniowaniem X (400 Gy) lub nienapromieniowane zawiesiny erytrocytów (Ht 2%) w buforowanej fosforanami soli fizjologicznej traktowano mannitolem (do 100 mM) lub etanolem (do 60 mM) i przechowywano w 37C do 72 godz. Określano hemolizę, kształt i granularność erytrocytów, profil elektroforetyczny białek błonowych oraz poziom utleniania hemoglobiny i glutationu.
Przeprowadzone badania wskazują, że zastosowane stężenia mannitolu hamują hemolizę erytrocytów, nie zapobiegając utlenianiu Hb i GSH. Mannitol zapobiegał nieznacznie pęcznieniu erytrocytów w stężeniach powyżej 20 mM. Etanol w stężeniach poniżej 60 mM nie wykazuje wpływu na poziom hemolizy erytrocytów, natomiast hamuje utlenianie Hb i GSH. Nie zaobserwowano znaczącego wpływu etanolu na wielkość erytrocytów. Jednak obserwuje się zwiększenie granularności komórek. Obserwowane różnice w działaniu badanych alkoholi na erytrocyty mogą wynikać z różnic w budowie chemicznej alkoholi oraz ich transporcie przez błonę plazmatyczną.
09/2008: pages 225-228; , ISBN: ISBN 978-83-926758-0-8
[Show abstract][Hide abstract] ABSTRACT: Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) by products of water radiolysis and by secondary radicals localized on haemoglobin (Hb) and human albumin (HSA) was studied.
Aqueous solutions of ADH, GAPDH and LDH were irradiated under air and under nitrous oxide (N2O) in the absence and in the presence of Hb or HSA. In order to determine the effectiveness of inactivation of the enzymes by radicals localized on Hb and HSA, the inactivation efficiency determined experimentally was compared with that calculated under assumption that only hydroxyl radicals are responsible for the enzyme inactivation.
In the absence of other proteins, under air, GAPDH showed the highest radiation sensitivity, followed by ADH and LDH. The sequence was reverse under anaerobic atmosphere. Oxygen increased considerably the inactivation of GAPDH and ADH. Secondary albumin and haemoglobin radicals brought about considerable inactivation of GAPGH and ADH. Albumin radicals (HSA) generated under N2O inactivated GAPDH and ADH more effectively than haemoglobin radicals (Hb). Under air, however, inactivation of GAPDH and ADH by haemoglobin peroxyl radicals was higher than by albumin peroxyl radicals. LDH was resistant to inactivation by haemoglobin and albumin radicals, and peroxides of these proteins.
In the light of these results and literature data, the observed differences in the effectiveness of inactivation of the dehydrogenases studied by secondary protein radicals depend on the amino acid residues present at the active site and in its close neighborhood and on the number of amino acid residues available on the protein surface.
International Journal of Radiation Biology 02/2008; 84(1):15-22. · 1.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H2O2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H2O2 was added to the mixture of metHb and ADH [the (metHb+ADH)+H2O2] system (ADH was present in the system since the moment of addition of H2O2 i. e. since the very beginning of the reaction of metHb with H2O2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H2O2 with metHb [the (metHb+H2O2)5 min+ADH] system. In the first case all the products of reaction of H2O2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H2O2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H2O2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.
Biochimica et Biophysica Acta 02/2007; 1774(1):86-92. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Doxorubicin (DOX) and idarubicin (IDA) are anthracycline antibiotics, widely used in human cancer treatment. The present study addressed the effects of these two drugs on lipid bilayer fluidity, protein conformation and microviscosity in erythrocytes from acute myeloid leukaemia patients, using electron spin resonance (ESR) spectroscopy and fluorescence measurements. Only DOX caused statistically significant changes in the parameters examined. Within 30 min of drug injection, changes were observed in the fluidity of the hydrophobic parts of the lipid bilayer and erythrocyte membrane protein conformation. These changes persisted for up to 24h. Analysis of the EPR Tempamine spectrum also showed that the microviscosity of the erythrocyte interior increased during the early stages of the drug effect. Idarubicin, in contrast, caused no identifiable change in any of the parameters studied and therefore seems to be safe for erythrocytes. We conclude that IDA is markedly less toxic than DOX to erythrocytes from acute myeloid leukaemia patients.
Cell Biology International 03/2006; 30(2):127-32. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.
Redox report: communications in free radical research 02/2006; 11(6):263-71. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8 x 10(12) dm(3) mol(-1) s(-1), and 1.6 x 10(12) dm(3) mol(-1) s(-1), respectively.
[Show abstract][Hide abstract] ABSTRACT: The effect of IDA and glutaraldehyde on the properties of human erythrocytes was examined by Electron Spin Resonance spectroscopy and fluorescence measurements. In this study glutaraldehyde was used as the agent linking the drug to the erythrocyte membrane. We have demonstrated that idarubicin (IDA) alone caused only negligible changes of the membrane fluidity. When IDA preincubated erythrocytes were treated with glutaraldehyde, the alterations in the fluidity were observed in the polar parts as well as in the deeper regions of the cell membrane. The incorporation of drug and glutaraldehyde into human erythrocytes also caused conformational alterations of membrane cytoskeletal proteins and changes in the internal viscosity of the cells. Our data suggest that glutaraldehyde in idarubicin-pretreated erythrocytes may potentiate the drug toxicity leading to significant perturbations in the organization of the plasma membrane lipids and proteins.
International Journal of Pharmaceutics 07/2001; 220(1-2):43-51. · 3.99 Impact Factor