Joseph Belagyi

Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy

The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min.

Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis

BackgroundThermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC).ResultsSETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 ± 0.7°C, 57.9 ± 0.7°C, 63.7 ± 1.0°C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66°C – 77°C.ConclusionAccording to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0 – 10.0°C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.

Effects of decreased specific glutathione reductase activity in a chromate-tolerant mutant of Schizosaccharomyces pombe

A chromate-tolerant mutant chr1-663T bearing a stable one-gene mutation and its parental strain 6chr+ were used to investigate the background of CrVI tolerance in the fission yeast Schizosaccharomyces pombe. The mutant chr1-663T displayed a significantly decreased specific glutathione reductase (GR) activity coded by the pgr1+ gene compared with its parental strain. Transformants of the mutant chr1-663T with a nonintegrative pUR18N vector expressing the pgr1+ gene exhibited the same CrVI sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in CrVI tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased CrVI-reducing and HO•-producing ability, which suggested an unbalanced oxidoreduction state of cells and partial complementation of the GR function. No mutation was found in the sequences of the pgr1+ and the pap1+ (transcriptional regulatory gene of GR) genes of the CrVI-tolerant mutant by sequence analysis.

The abc1−/coq8− respiratory-deficient mutant of Schizosaccharomyces pombe suffers from glutathione underproduction and hyperaccumulates Cd2+

The abc1−/coq8− gene deletion respiratory-deficient mutant NBp17 of fission yeast Schizosaccharomyces pombe displayed a phenotypic fermentation pattern with enhanced production of glycerol and acetate, and also possessed oxidative stress-sensitive phenotypes to H2O2, menadione, tBuOOH, Cd2+, and chromate in comparison with its parental respiratory-competent strain HNT. As a consequence of internal stress-inducing mutation, adaptation processes to restore the redox homeostasis of mutant NBp17 cells were detected in minimal glucose medium. Mutant NBp17 produced significantly increased amounts of O2•− and H2O2 as a result of the decreased internal glutathione concentration and the only slightly increased glutathione reductase activity. The Cr(VI) reduction capacity and hence the •OH production ability were decreased. The mutant cells demonstrated increased specific activities of superoxide dismutases and glutathione reductase (but not catalase) to detoxify at least partially the overproduction of reactive oxygen species. All these features may be explained by the decreased redox capacity of the mutant cells. Most notably, mutant NBp17 hyperaccumulated yellow CdS.

Effects of hexavalent chromium on the plasma membranes of sensitive and tolerant mutants of Schizosaccharomyces pombe. An EPR study

The interactions of chromium(VI) with the plasma membranes of chromium-sensitive (chr-51S) and chromium-tolerant (chr1-66T) mutants and their parental strain (6chr(+)) of a Schizosaccharomyces pombe strain were studied by electron paramagnetic resonance (EPR) spectroscopy. 5-doxylstearic acid (5-SASL) and 3-doxylbutyric acid (HO-185) spin probes were used to label the membranes. The order parameter S from the EPR spectra was calculated at different temperatures (0-25 degrees C) in order to characterize the internal dynamics of the membranes. In control experiments, both mutants exhibited differences in structural transitions in the both 5-SASL- and the HO-185-labeled membranes in comparison with their parental strain, suggesting differences in the membrane composition and/or rotational dynamics of these mutants. Addition of K(2)Cr(2)O(7) (225 microM) induced small decreases in the phase transition temperatures of the 5-SASL-labeled membranes of the parental and chromium-sensitive strains. More pronounced effects of the chromium compound on the HO-185-labeled membranes were detected as evidence that the membrane perturbations are mostly localized in the environment of the lipid-water interface.

Nucleotide-induced changes in muscle fibres studied by DSC and TMDSC

Abstract Differential scanning calorimetry (DSC) was used in conventional and temperature-modulated mode to study the energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were used in the experiments with and without the presence of nucleotides (ATP, ADP, AMP–PNP) and ATP or ADP+orthovanadate. In the complex DSC pattern, the higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region. The monoton decreasing TMDSC heat capacities show that the C p of biological samples should not be temperature independent.

Pro-oxidative versus antioxidative reactions between Trolox and Cr(VI): The role of H2O2

The effect of the Vitamin E model compound Trolox in reactions with Cr(VI) in the presence or absence of hydrogen peroxide was investigated. The aim of this study was to establish and discuss potential Trolox-mediated pro-oxidative reactions. The importance of the Trolox:Cr(VI) ratio in the Cr(VI) reduction process was determined from the EPR spectra and DNA cleavage reactions. In the absence of hydrogen peroxide, reduction of Cr(VI) occurred with concomitant oxidation of Trolox to the phenoxyl radical. In the presence of hydrogen peroxide, Cr(V), produced by the reduction of Cr(VI), reduced hydrogen peroxide to the hydroxyl radical. The latter was detected by spin-trapping the methyl radical following reaction with N-methyl sulfoxide. During Cr(VI) reduction with Trolox, DNA single- or double-strand breaks due to Trolox radical formation were not observed. Relaxed DNA appeared only when H(2)O(2) was added to Trolox/Cr(VI) mixtures most probably due to hydroxyl radical formation during the redox cycling of Cr(V/IV)-species. Fenton-like reactions do not play a significant role in the Trolox/Cr(VI) system in the absence of added H(2)O(2).

Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

Mild oxidation of –SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules.

Effect of polycyclic aromatic hydrocarbons on erythrocyte membranes by DSC and EPR

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on human erythrocyte membranes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model systems in order to study the effect the polycyclic aromatic hydrocarbons on lipid structure and dynamics. Eight different compounds among others naphthalene and pyrene were compared, which occur in significant concentrations in dust collected from the air in large cities. Experiments using spin label technique showed that the compounds induced mobility changes in the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes. The effects depended on the structure and concentration of the different compounds. Similarly to EPR observations, DSC measurements reported decrease of transition temperature in comparison to control DPPC vesicles. These results suggest that polycyclic aromatic hydrocarbons were able to modify the internal dynamics of erythrocyte membranes which might lead to damage of the biological functions.

UV generated oxygen free radicals in cardiac myosin. DSC and EPR study

Abstract Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were used to study the motional dynamics and segmental flexibility of cardiac myosin in the presence of free radical generating system. Cardiac myosin isolated from calf heart muscle were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules attached to cardiac myosin decreased with time following a single exponential curve. MgADP and MgADP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. DSC measurements resulted in two main transitions at 49.4°C and 54.1°C, respectively. Addition of MgADP produced a decrease of 49.4°C transition, whereas a shift towards higher temperature was detected at 54.1°C transition. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. The total enthalpy of the UV irradiated myosin decreased significantly in different intermediate states of the ATP cycle.