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1.
Thermal stability and internal dynamics of myosin head in psoas muscle fibres of rabbit in the intermediate state AM.ADP.Pi - mimicked by AM.ADP.Vi - of the ATP hydrolysis cycle was studied by differential scanning calorimetry and spin label electron paramagnetic resonance spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding ADP and weakly binding AM.ADP.Vi state of myosin to actin. The transition at 54.0°C can be assigned to the 50 k actin-binding domain. The transition at highest temperature (67.3°C) represents the unfolding of actin and the contributions arising from the nucleotide-myosin head interaction. The transition at 58.4°C reflects the melting of the large rod part of myosin. Nucleotide binding (ADP, ATP plus orthovanadate) induced shifts of the melting temperatures and produced changes in the calorimetric enthalpies. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads in agreement with DSC findings. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

2.
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   

3.
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   

4.
The internal dynamics and the thermal stability of myosin in rabbit psoas muscle fibres in different intermediate states of the ATP hydrolysis cycle were studied by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding and weakly binding state of myosin to actin. The transition at 58.4°C can be assigned to the nucleotide-binding domain. The transition at highest temperature represents the unfolding of the actin and the contributions arising from the actin-myosin interaction. The transition of 54°C reflects the interaction between the subunits of myosin. Nucleotide binding induced shifts of the melting temperatures and produced variations in the calorimetric enthalpy changes. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

5.
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution 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 myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

6.
Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins.Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns.DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.This work was supported by grants from the National Research Foundation (OTKA T 017099) and Ministry of Social Welfare (ETT 737/1993). The Bruker ESP 300 E spectrometer and the SETARAM Micro DSC-II used in the experiments were purchased with funds provided by the National Research Foundation Grants CO-123 and CO-272. The computer (PC-386) was supported by POPEX Ltd., Pécs, Hungary. The authors thank to Prof. Dr. K. Hideg (Central Research Laboratory) for providing the iodoacetamide spin label.  相似文献   

7.
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   

8.
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   

9.
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   

10.
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   

11.
In this paper, I will sum up our research activity from this field performed in the last about 25 years. I will focus on three main points: basic muscle research in the different intermediate states of ATP hydrolysis cycle during muscle contraction, R&D activities to develop and test different dairy products and TA application in some surgical and diagnostic problem. Our initial research concerned the investigation of thermal stability of main muscle proteins clarifies their basic unfolding characteristics. Later we extended the thermal stability investigation from protein solution to the myosin myofibrils and to the higher organization of muscle proteins, the muscle fibres, checking the effect of nucleotides. At that time, it became possible to stabilize the different intermediate states of ATP hydrolysis up to the time of DSC measurement, using different Pi analogues (Vi, AlFx and BeFx) and non-hydrolysable ATP analogue (AMP.PNP). This way the targets were AM.ADP.Vi (and with AlFx or BeFx) so-called weak binding state, AM.ADP the “strong” binding state as well as the “rigor” AM complex state. AM.AMP.PNP state was used to mimic the AM.ATP state. With our R&D cooperation, a cold spreadable butter was successfully developed. We were a partner in the development of Ca-enriched cheese, in its spreadable form too as well as in the development and testing of different dairy products using probiotic cultures. Our TA activity covers a wide range of medical applications, e.g. investigation of the different abnormalities of human leg skeletal muscle, different stages of degeneration of human vertebral disc, to judge the goodness/applicability of different suture technique on tracheal cartilage in primary airway reconstruction or the characterization of different self-expandable stents implantation in the oesophagus treatment. We have joined those groups who try to use DSC in the diagnosis of different diseases from blood plasma, e.g. in the case of breast cancer, melanoma, in psoriasis.  相似文献   

12.
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   

13.
Summary DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure, but the protein system as well. These might lead to further damage of the biological functions.  相似文献   

14.
We conducted 31P NMR kinetic studies and 1H-diffusion measurements on myosin-catalyzed hydrolysis of adenosine triphosphate (ATP) under varied conditions. The data elucidate well the overall hydrolysis rate and various factors that significantly impact the reaction. We found that the enzymatic hydrolysis of ATP to adenosine diphosphate (ADP) was followed by ADP hydrolysis, and different nucleotides such as ADP and guanosine triphosphate acted as competitors of ATP. Increasing ATP or Mg2+ concentration resulted in decreased hydrolysis rate, and such effect can be related to the decrease of ATP diffusion constants. Below 50 °C, the hydrolysis was accelerated by increasing temperature following the Arrhenius’ law, but the hydrolysis rate was significantly lowered at higher temperature (~60 °C), due to the thermal–denaturation of myosin. The optimal pH range was around pH 6–8. These results are important for characterization of myosin-catalyzed ATP hydrolysis, and the method is also applicable to other enzymatic nucleotide reactions.  相似文献   

15.
Actin is one of the important elements of the striated muscle that transmits force from the myosin filaments and as a part of the cytoskeleton plays an important role in shape determination of cells. It is a known experience that removal of the divalent cation affects the dynamic behaviour of actin in both forms. Paramagnetic probes and electron paramagnetic resonance (EPR) spectroscopy provide direct technique by which the rotation and the orientation of specifically labelled proteins can be followed during biochemical manipulations. The spectroscopic measurements could be combined with DSC measurements that report domain stability and interactions and allow the calculation of the thermodynamic parameters during the melting process. Actin was spin-labelled with maleimide or fluoro-dinitro proxyl probe molecules which are bound to the Cys-374 or Lys-61 residues of the smaller domain. EPR spectroscopy spectra were recorded in monomer form in Ca- and EGTA-state as a function of temperature up to the melting point. Similarly, DSC measurements were performed and analyzed using the kinetic theory. The measurements showed that removal of the divalent cation from the globular actin induced significant local and global structural change both in the thermodynamic properties and the rotational mobility of actin detected by DSC and EPR. On the basis of the results derived by deconvolution of the DSC pattern we can suggest a non-interactive two-domain melting for the monomer actin after removing the divalent cations.  相似文献   

16.
The functional transitions of the membrane-bound chloroplast ATPase (CF1) as influenced by low ADP and uncoupler concentrations are investigated by measurements of initial and steady-state ATP hyrolysis and concomitant membrane energization. Following activation of latent ATP hydrolysis by light in the presence of dithioerythritol, the resulting steady-state ATP hydrolysis depends on the dark-period ( t d) bteween light activation and ATP addition. ADP, added during t d, inhibits this activity ( K i about 2 μ M ) and induces a lag in the onset of ATP hydrolysis. The extent of membrane energization as monitored by an aminoacridine fluorescent probe is proportional to the ATPase activity.
An uncoupler amplifies the inhibitory effect of ADP if added during f d, whereas it induces the normal stimulation of ATP hydrolysis in the absence of ADP. The ADP effect, which is different from product inhibition, is interpreted as a conformational interaction with CF1 causing an increase of the energy threshold required for the inactive → active transition of the CF1 molecules. These results are in harmony with currently proposed models of CF1 regulation by adenine nucleotides based on binding studies.
The inactive → active transition of CF1 conformation is investigated by analysis of the lag in the onset of ATP hydrolysis at different ADP concentrations and by means of varied light pulses and single-turnover flashes, using the electric potential indicating absorption change at 515 nm as a probe for the onset of ATP hydrolysis. The half-time of the process leading to fully (re)activated ATP hydrolysis is about 0.25 s. The ATP-dependent flash-induced inactive → active transition occurs within a few turnovers of electron flow.  相似文献   

17.
EPR evidence is presented to support the literature contention that a small spherical probe will detect chain melting while a longer probe will detect the glass transition of MBBA. One finds DTA/DSC values for Tg from both probes when using the Grest-Cohen model near room temperature.  相似文献   

18.
We employ temperature modulated DSC (TMDSC) to determine the dependence of the fictive temperature on cooling rate for a series of polychlorinated biphenyls (PCB). From the slopes of semi-logarithmic plots of cooling rate vs. fictive temperature, the latter normalized by the fictive temperature for an arbitrary cooling rate, we determine the enthalpic fragilities. Despite significant differences in glass transition temperature and chemical structure (specifically chlorine content), the PCB have the same fragility. The value of the fragility determined using TMDSC is consistent with the fragility previously determined using dielectric relaxation spectroscopy.  相似文献   

19.
The definition of the glass transition temperature, T g, is recalled and its experimental determination by various techniques is reviewed. The diversity of values of T g obtained by the different methods is discussed, with particular attention being paid to Differential Scanning Calorimetry (DSC) and to dynamic techniques such as Dynamic Mechanical Thermal Analysis (DMTA) and Temperature Modulated DSC (TMDSC). This last technique, TMDSC, in particular, is considered in respect of ways in which the heterogeneity of the glass transformation process can be quantified.  相似文献   

20.
Blends of poly(ether ketone) (PEK) with poly(terephthaloyl-imide) (a thermoplasticpolyimide, TPI) were studied by temperature-modulated DSC (TMDSC) and X-ray diffraction. Samples were prepared by compression moulding of the premixed materials at 400°C and quenched to prevent crystallisation.The amorphous blends showed a single glass transition but with a jump in the temperature value at 60 mass% of PEK, indicating limited miscibility of the system at both sides of the composition series in the quenched, glassy state. Two cold crystallisation peaks over the concentration range 30 to 70 mass% of PEK were observed, but only one for all other compositions. A single melting peak was observed in all systems.Blends crystallised from the glassy state showed eutectic behaviour with the presence of the crystals of both pure components. This is the first reported case of two semicrystalline polymers exhibiting eutectic co-crystallisation. The formation of eutectic crystals is proof of full miscibility of the two polymers in their liquid state, i.e. at a temperature of 400°C and above. Blends cooled from the melt at a cooling rate of 2 K min–1 showed a single glass transition and an extended melting range.Crystallisation during a second melting run generally starts at a different temperature then during the first run indicating chemical changes occurred in the molten state. This change was also verified by an exothermic peak above the melting temperature using TMDSC.This revised version was published online in November 2005 with corrections to the Cover Date.  相似文献   

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