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1.
The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK. 相似文献
2.
Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state 总被引:1,自引:0,他引:1
Ou Wb Wang RS Lu J Zhou HM 《The international journal of biochemistry & cell biology》2002,34(8):970-982
The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding. 相似文献
3.
The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCI, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding. 相似文献
4.
The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region. 相似文献
5.
The role of disulfide bonds in the conformational stability and catalytic activity of phytase. 总被引:3,自引:0,他引:3
Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity. 相似文献
6.
The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation. 相似文献
7.
Mu H Zhou SM Yang JM Meng FG Park YD 《International journal of biological macromolecules》2007,41(4):439-446
The dimeric native state of creatine kinase (CK) was aggregated at conspicuous levels during cysteine modification at the active site with using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) under a high enzyme concentration. Measuring the ANS-binding fluorescence revealed that the hydrophobic surface of CK was increased by cysteine modification due to the flexible active site, and this resulted in insoluble aggregation, probably via non-specific hydrophobic interactions. To determine whether the aggregates can be refolded, 3M guanidine hydrochloride (GdnHCl) was used to dissolve the aggregates into the denatured form. Refolding of the solubilized enzyme sample was then conducted, accompanied by deprivation of DTNB from the CK in the presence of DTT. As a result, CK was reactivated by up to 40% with partial recovery of the tertiary (78%) and secondary structures (77%). To further elucidate its kinetic refolding pathway, both time interval measurements and a continuous substrate reaction were performed. The results showed that the refolding behavior was similar to the manner of normal CK folding with respect to the following two-phase kinetic courses. Additionally, the rate constants for the dimerization of the unfolded CK were dependent on the enzyme concentration and this was irrespective to the DTT concentrations, suggesting the rate-limiting steps of CK reassociation. The present study will expand our insight into the flexibility of the enzyme active site, which might act as a risk factor for inducing the unfavorable aggregation and partial refolding pathway of CK, as well as inducing an intermediate-like state recovery from aggregation. 相似文献
8.
Mariana N Dimitrova Roman H Szczepanowski Sergei B Ruvinov Alan Peterkofsky Ann Ginsburg 《Biochemistry》2002,41(3):906-913
The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI. 相似文献
9.
The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in different concentrations of trifluoroethanol (TFE) have been investigated by far-ultraviolet circular dichroism and fluorescence emission spectra. Unfolding and activation rate constants were measured and compared, the activation and inactivation courses were much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole. However, compared to other metalloenzymes, CIP is inactivated at higher concentrations of TFE as denaturant. 相似文献
10.
Kuznetsova IM Stepanenko OV Turoverov KK Zhu L Zhou JM Fink AL Uversky VN 《Biochimica et biophysica acta》2002,1596(1):138-155
GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of "phase diagram", i.e. I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are fluorescence intensity values measured on wavelengths lambda(1) and lambda(2) under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated. 相似文献
11.
alpha-Amylase from mung beans (Vigna radiata) being one of the few plant alpha-amylases purified so far was studied with respect to its conformational stability by CD and fluorescence spectroscopy. The enzyme was shown to bind 3-4 Ca(2+) ions, which all are important for its activity. In contrast to other alpha-amylases no inhibition was observed at high Ca(2+) concentrations (100 mM). Depletion of calcium decreased the transition temperature from 87 to 48 degrees C. Kinetic stopped-flow fluorescence measurements allowed detecting two unfolding phases at >6 M GdmCl, whereas only one phase was observed at <5 M GdmCl. These results suggest that the first (reversible) step of unfolding is slower than the second (irreversible) step at low GdmCl concentrations, whereas the rates of these two steps are opposite at high GdmCl concentrations. 相似文献
12.
The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding. 相似文献
13.
The inactivation and conformational changes of the bacterial chaperonin GroEL have been studied in SDS solutions with different concentrations. The results show that increasing the SDS concentration caused the intrinsic fluorescence emission intensity to increase and the emission peak to slightly blue-shift, indicating that increasing the SDS concentration can cause the hydrophobic surface to be slightly buried. The changes in the ANS-binding fluorescence with increasing SDS concentration also showed that the GroEL hydrophobic surface decreased. At low SDS concentrations, less than 0.3 mM, the GroEL ATPase activity increased with increasing SDS concentration. Increasing the SDS concentration beyond 0.3 mM caused the GroEL ATPase activity to quickly decrease. At high SDS concentrations, above 0.8 mM, the residual GroEL ATPase activity was less than 10% of the original activity, but the GroEL molecule maintained its native conformation (as indicated by the exposure of buried thiol groups, electrophoresis, and changes of CD spectra). The above results suggest that the conformational changes of the active site result in the inactivation of the ATPase even though the GroEL molecule does not markedly unfold at low SDS concentrations. 相似文献
14.
The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5-25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25-40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The alpha-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions. 相似文献
15.
Inactivation and conformational changes of creatine kinase at low concentrations of hexafluoroisopropanol solutions. 总被引:1,自引:0,他引:1
Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%-5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%-2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate-enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole. 相似文献
16.
Xie Q Guo T Lu J Zhou HM 《The international journal of biochemistry & cell biology》2004,36(2):296-306
Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway. 相似文献
17.
Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion. 相似文献
18.
A mutant of dimeric rabbit muscle creatine kinase (CK), in which six residues (residues 2-7) at the N-terminal were removed by the PCR method, was studied to assess the role of these residues in dimer cohesion and to determine the structural stability of the protein. The specific activity of the mutant was 70.39% of that of the wild-type CK, and the affinity for Mg-ATP and CK substrates was slightly reduced compared with the wild-type protein. The structural stability of the mutant was investigated by a comparative equilibrium urea denaturation study and a thermal denaturation study. The data acquired by intrinsic fluorescence and far-UV circular dichroism (CD) during urea unfolding indicated that, the secondary and tertiary structures of the mutant were more stable than those of wild-type CK. Furthermore, results of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence demonstrated that the hydrophobic surface of the mutant CKND(6) was more stable during urea titration. Data from size exclusion chromatography (SEC) experiments indicated that deletion of the six N-terminal residues resulted in a relatively loose molecular structure, but the dissociation of the mutant CKND(6) occurred later during the unfolding process than for wild-type CK. Consistent with this result, the differential scanning calorimetry (DSC) profiles demonstrated that the thermal stability of the enzyme was increased by removal of the six N-terminal residues. We conclude that a more stable quaternary structure was obtained by deletion of the six residues from the N-terminal of wild-type CK. 相似文献
19.
氨基酰化酶在阴离子去圬剂十二烷基硫酸锂(LDS)溶液中的失活与去折叠的研究结果表明,在低浓度的LDS溶液(0.6mmol/L)中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化。而酶的活力已经大部分或几乎全部丧失。当LDS浓度达1.6mmol/L时,此时酶分子的构象变化才达到最大程度,在实验使用的LDS的浓度范围内,用远紫外CD光谱监测的二级结构没有发生明显的变化。从上述研究结果,可以认为含锌氨基酰化酶的活性部位也具有相对的柔性。 相似文献
20.
谷胱甘肽磷脂氢过氧化物酶是唯一能够直接还原生物膜上脂类过氧化物的过氧化物酶.本文利用圆二色光谱(CD)、内源荧光光谱和差示扫描量热仪(DSC)研究了温度对谷胱甘肽磷脂氢过氧化物酶(OsPHGPx)活性及其构象变化的影响.在温度为 20-27.5℃ 期间,随着温度的逐渐升高,OsPHGPx 的活性逐渐上升,到 27.5℃ 时达到最大值;在 27.5-45℃ 时,随着温度逐渐升高,其活性迅速下降;当温度超过45℃时,其活性完全尚失. 在20-40℃,CD 光谱、内源荧光光谱和 DSC 均没有发生明显变化,暗示OsPHGPx 的结构基本保持完整;在 40-55℃,CD 光谱显示该酶二级结构发生去折叠;内源荧光光谱的变化暗示该酶三级结构发生去折叠.其中在40-45℃,DSC显示该酶可能存在两个去折叠中间体.当温度超过 55℃ 时,整个酶的构象不再发生变化,呈现去折叠状态. 相似文献