共查询到20条相似文献,搜索用时 31 毫秒
1.
Lőrinczy D. Gaszner B. Könczöl F. Belágyi J. 《Journal of Thermal Analysis and Calorimetry》2000,61(2):597-605
Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation
transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence
of free radical generating system.
Muscle fibre bundles isolated from psoas muscle of rabbit 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 decreased with time following a single exponential curve. MgADP and MgATP 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. The analysis of the EPR spectra of spin-labelled and oriented fibres
showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by
computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the
expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding
state.
DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition
of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C
transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced
further shifts of the transition temperatures and affected the width of the heat absorption curves.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
Lőrinczy D. Könczöl F. Farkas L. Belagyi J. Schick C. 《Journal of Thermal Analysis and Calorimetry》2001,66(2):633-644
Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature
modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin
ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive
sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or
ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP
state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population
of spin labels was superimposed on the ADP-like spectrum.
In the complex DSC pattern, 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 C
p of biological samples should not be temperature independent.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
3.
4.
Intermediate states of myosin head during atp hydrolysis cycle in psoas muscle fibres by EPR and DSC
Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle,
myosin and actin. The process is driven by the energy liberated from the hydrolysis of ATP. In the presence of CaATP the energy
released from hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion
of myosin head while bound to actin. It is suggested that myosin heads attached to actin produce conformational changes during
the hydrolysis process of ATP, which results in a strain in the head portion of myosin in an ATP-dependent manner. These structural
changes lead to a large rotation of myosin neck region relieving the strain.
Paramagnetic probes and EPR spectroscopy provide direct method in which the rotation and orientation of specifically labelled
proteins can be followed during muscle activity. In order to find correlation between local and global structural changes
in the intermediate states of the ATPase cycle, the spectroscopic measurements were combined with DSC measurements that report
domain stability and interactions. 相似文献
5.
Lőrinczy D. Hartvig N. Farkas N. Belagyi J. 《Journal of Thermal Analysis and Calorimetry》2001,65(2):351-358
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres
to study the effect of the binding of nucleotides (ADP and AMP⋅PNP) to myosin. The thermal unfolding of muscle fibres showed
three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMP⋅PNP markedly affected the transitions,
implying the strong interaction between AMP⋅PNP and catalytic domain, and partial dissociation of heads from actin. ADP produced
only small changes in transition temperatures.
Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMP⋅PNP-state resulted in two populations; 50% of labels
was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The conformation of
the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin
differ from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state
to actin might have different local conformations.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
6.
Selective purification of cardiac myosin by a high-performance salicylate affinity column 总被引:1,自引:0,他引:1
M W Strohsacker M D Minnich M A Clark R G Shorr S T Crooke 《Journal of chromatography. A》1988,435(1):185-192
The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin. 相似文献
7.
T. Dergez F. Könczöl N. Farkas J. Belőgyi D. Lõrinczy 《Journal of Thermal Analysis and Calorimetry》2005,80(2):445-449
Summary The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·Vi, ADP·AlF4) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were Tm1=52.9±0.7°C, Tm2=57.9±0.7°C, Tm3=63.7±1.0°C and Tm4=67.8±0.7°C, but the last transition increased to higher temperature depending on the Pi analogue. 相似文献
8.
The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin
label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong
as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different
stability (T
m =54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at T
m =58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly
(T
m =70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres
(labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin.
This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres
AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like
spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported
increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence
of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational
change.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
Piotr Dobryszycki Dorota Krzyzanowska Marian Kochman 《Journal of Chemical Sciences》1991,103(3):435-440
The range of flexibility of the rabbit muscle aldolase molecule was studied using fluorescent labelled aldolase. The protein
molecule was specifically labelled on the opposite sites of the enzyme subunit with the fluorescence energy donor and acceptor
residues. Labelled aldolase with full enzymatic activity was used as a tool in the FRET studies between IAEDANS — donor or
Cys-289 and IAF — acceptor on Cys-239. A range of Forster distance (R) was obtained by collisional quenching of the donor emission. The experiments of donor fluorescence quenching with wide range
of acrylamide concentrations have shown the changes of donor-acceptor distances. In the absence of quencher the D-A distance
distribution in characterized by an average value of 40.4 ?, and a half-width of 0.13 ?. A dramatic increase in half-width
to 17.7? is observed after expositions of the enzyme to high acrylamide concentrations (0.13 M-0.68 M). 相似文献
10.
K. Türmer F. Könczöl D. Lőrinczy J. Belágyi 《Journal of Thermal Analysis and Calorimetry》2012,108(1):95-100
Actin is the component of several biological systems and it plays important role in different biological processes, especially
in cell motility. The actin-based motility is accompanied with ATP-consume, and the irreversible ATP hydrolysis is coupled
with the polymerization of monomer actin into filamentous form. When an actin monomer is incorporated into a filament, the
ATPase is activated, and thereby the polymer formation is promoted. The polymer formation and the ATP hydrolysis is associated
with internal motions and significant changes of the conformation in reaction partners. In this article, the ATP nucleotide
in monomer actin was exchanged by its non-hydrolyzable analogue adenylyl-imidodiphosphate (AMP.PNP), and using two biophysical
methods, electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC), we studied the local
and global changes in globular and fibrous actin following the nucleotide exchange. The paramagnetic probe molecule—a maleimide
spin label—was attached to Cys-374 site of monomer actin, and its rotational mobility was derived at different temperature.
In DSC measurements the transition temperatures of samples with different bound nucleotides were compared. From the measurements
we could conclude, that the nucleotide exchange induces changes in the internal rigidity of the actin systems, AMP.PNP-actins
showed longer rotational correlation time and increased thermal transition temperature. 相似文献
11.
J. Orbán Sz. Halasi G. Papp Szilvia Barkó Beáta Bugyi 《Journal of Thermal Analysis and Calorimetry》2005,82(1):287-290
Summary The thermodynamic properties of the cardiac and skeletal a-actin isoforms were studied to characterize the molecular bases
of the functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties
of the actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free
energy changes the α-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated
form. The magnesium saturated form of the α-cardiac actin filaments seemed to be more stable compared to the calcium saturated
form of it. The enthalpy and entropy changes could differentiate between the α-cardiac and α-skeletal actin isoforms and between
the calcium and magnesium saturated cardiac actin isoforms as well. Our results can demonstrate that the few differences between
the amino acid sequences of the α-actin isoforms have an influence on the thermal properties and maybe on the function of
these proteins as well. 相似文献
12.
We have chosen 3-ethoxypropan-1-ol (EtOPrOH) as a typical short-carbon-chain ether–alcohol used as industrial solvent and
have analyzed the degradation products resulting from its attack by •OH radicals generated by the photo-Fenton reaction. The laboratory conditions were representative of those found in tropospheric
water droplets. Twelve products have been identified by use of GC–MS analysis, either directly or after extraction by SPME
fibers, and by HPLC–UV analysis with a special column for carboxylic acids and after reaction of carbonyl groups with 2,4-dinitrophenylhydrazine. These products contain one to three carbon atoms (instead of five in EtOPrOH), among which one or
two are oxidized. According to the reaction pathways proposed, seven products—including methanal—can result from attack by
one •OH only, three products imply attack by a second •OH, as expected from their higher oxidation number, and it is suggested that reaction between two organic radicals is needed
for formation of only two products. The relevance of this investigation to the fate of EtOPrOH and similar ether–alcohols
in the troposphere is briefly discussed. 相似文献
13.
Barbara Marciniec Katarzyna Dettlaff Marek Naskrent Zuzanna Pietralik Maciej Kozak 《Journal of Thermal Analysis and Calorimetry》2012,108(1):33-40
The effect of ionising radiation on the physico-chemical properties of disulfiram (Antabuse, Esperal, bisdiethylthiocarbamoil
disulphide) has been studied by DSC, FTIR, EPR, MS, TLC and HPLC. Sterilisation was carried out in the solid state, at room
temperature and normal air humidity using the electron beam of 9.96 Mev from accelerator. All the measurements were made simultaneously
for the irradiated and nonirradiated substance. It has been found that the drug studied in solid phase when subjected to an
electron beam corresponding to the irradiation in the doses 10–100 kGy shows the presence of free radicals (EPR), and a change
in colour from white to pale green-grey that disappears after solution in water or methanol. After the irradiation with the
dose of 100 kGy, its melting point and enthalpy slightly decreased. Also the content of the active substance decreases (HPLC
−1.5%, UV −3.6%, iodometric titration method −2.7%) and trace amounts of the radiolysis products appear (HPLC). The substance
irradiated with the doses 10–50 kGy does not show changes in the melting point, in the content and presence of the radiolysis
products. The EPR results have shown that free radicals disappear after about a year and the discolouring disappears with
them. The results of this study have shown that disulfiram can be subjected to sterilisation by irradiation with no deterioration
of its physico-chemical properties, but a long time of storage needed to remove free radicals and discolouration questions
the economic justification for this type of sterilisation. 相似文献
14.
JPC – Journal of Planar Chromatography – Modern TLC - The discovery of the implication of free radicals in the etiology of many diseases has led to increased interest in functional food... 相似文献
15.
O. V. Drygina V. B. Panov O. Yu. Okhlobystin 《Chemistry of Heterocyclic Compounds》1980,16(2):130-133
The corresponding o-carboranylpyrylium perchlorates were obtained from 4H-pyrans of the o-carborane series under the influence
of 70% HClO4 in acetic anhydride or 2,2′,4,4′-tetramethoxydiphenylammonium perchlorate. The carboranylpyranyl free radicals were studied
by EPR spectroscopy, and it was shown that the unpaired electron is localized in the carborane ring.
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 185–188, February, 1980. 相似文献
16.
B. L. Tumanskii E. N. Shaposhnikova O. G. Kalina V. V. Bashilov M. N. Nefedova S. P. Solodovnikov N. N. Bubnov V. I. Sokolov 《Russian Chemical Bulletin》2000,49(5):843-846
The influence of structurally different methanofragments attached to [60]fullerene on the spectral parameters of the spin-adducts
of phosphoryl radicals with these compounds was studied by ESR spectroscopy. The main direction of attack of phosphoryl radicals
is the same fullerene hemisphere to which the methanofragment is attached.
Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya. No. 5, pp. 845–848, May, 2000. 相似文献
17.
Dolenc J Riniker S Gaspari R Daura X van Gunsteren WF 《Journal of computer-aided molecular design》2011,25(8):709-716
Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor,
which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of
receptor flexibility to relative binding free enthalpies, two host–guest systems have been examined: inclusion complexes of
α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the
minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations
reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand–receptor
binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced
in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand–DNA
binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand–DNA
interactions but also from changes in ligand–solvent interactions as well as from the loss of DNA configurational entropy
upon restraining. 相似文献
18.
Substituent effects on the structure of radicals and parent hydrocarbons formed by isolated or condensed three-membered rings
have been investigated by Hartree-Fock, post-Hartree-Fock and density functional methods. The trends of structural parameters
computed for the hydrocarbon systems are in agreement with available experimental data. Substituent effects can be rationalized
in terms of interactions between localized orbitals obtained by natural bond analysis. The effects are even larger in free
radicals and can be analyzed using the same model.
Received: 13 March 1998 / Accepted: 13 July 1998 / Published online: 9 October 1998 相似文献
19.
We have examined by DSC the complexes of myosin with actin in fibre system in the absence of nucleotides and the intermediate state of ATP hydrolysis by mimicking stable complex with myosin and ADP and beryllium fluoride in muscle fibres. Comparing the DSC results with other structural analogues of phosphate Pi leads the conclusion that the AM.ADP.BeFx complex favours the AM.ADP.Pi complex in fibre system. The deconvolution of DSC scans resulted in four transitions, the first three transition temperatures were almost independent of the intermediate state of the muscle, the last transition temperature was shifted to higher temperature, depending on the actual intermediate states of ATP hydrolysis. In AM.ADP.Vi state the transition temperature at the second and third transitions (actin binding domain and myosin rod) varied only slightly, whereas the last one (the fourth transition) shifted markedly to higher temperature depending on the ternary complex, e.g. in case of ADP plus BeFx it was 77.7 °C, the highest value in weakly binding state of myosin to actin. The sum of calorimetric enthalpies of the first and last curves was practically constant, but their fractions depended on the state of the muscle. In strongly binding state of myosin to actin (rigor, ADP state) the fraction of the first transition was much larger, than the last one, whereas in weakly binding state of myosin to actin, the fraction of the first transition decreased at the expense of the last one. It supports also the view that these transitions are parts of the same portion of the myosin molecule. 相似文献
20.
Kinetics of hydrogen peroxide decomposition in the presence of the tetraaquapalladium(II) complex in an aqueous solution at
40–70 °C was studied. The reaction rate is the first order with respect to the concentration of both PdII and H2O2 and the negative first order with respect to perchloric acid. Using free radicals traps, the reaction mechanism was found
to differ from the traditional free-radical mechanism known for d-metal aqua ions and proceeds without generation of hydroxyl
radicals. The kinetic data obtained suggest a mechanism involving the formation of an intermediate palladium complex with
oxygen.
__________
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1077–1082, May, 2005. 相似文献