共查询到20条相似文献,搜索用时 62 毫秒
1.
Vitaly N. Nikandrov Oleg N. Murashko Galina V. Vorobyova Nelly S. Pyzhova Natalie V. Kvyatkovskaya Oksana A. Bartalevich 《International journal of peptide research and therapeutics》1997,4(4-6):497-502
Summary The formation of stable equimolar complexes of streptokinase or plasminogen with muscle lactate dehydrogenase or pyruvate
kinase, heart mitochondrial malate dehydrogenase and hepatic catalase at pH 7.4, 3.0 and 10.0 was first detected by differential
spectroscopy methods. All complexes, except those of plasminogen with dehydrogenases, were resistant to 6 M urea. Judging
from circular dichroism spectra, tertiary and secondary structures were considerably changed in the complexes. These changes
were significantly dependent upon the nature of interacting proteins; in some cases their structures were more ordered. NAD
(but not NADH) hampered the formation of streptokinase complexes with dehydrogenases. The plasminogen-activating function
of streptokinase and the ability of plasminogen to be activated by streptokinase in the complexes with oxidoreductases were
essentially unchanged. Pyruvate kinase induced a moderate (by 35%) increase in the streptokinase activating function. It is
assumed that the formation of complexes of streptokinase or plasminogen with enzymes may serve as a link in metabolic regulation
and/or intercellular interactions. 相似文献
2.
Vitaly N. Nikandrov Oleg N. Murashko Galina V. Vorobyova Nelly S. Pyzhova Natalie V. Kvyatkovskaya Oksana A. Bartalevich 《Letters in Peptide Science》1997,4(4-6):497-502
The formation of stable equimolar complexes ofstreptokinase or plasminogen with muscle lactatedehydrogenase or pyruvate kinase, heart mitochondrialmalate dehydrogenase and hepatic catalase at pH 7.4,3.0 and 10.0 was first detected by differentialspectroscopy methods. All complexes, except those ofplasminogen with dehydrogenases, were resistant to 6 Murea. Judging from circulardichroism spectra, tertiary and secondary structureswere considerably changed in the complexes. Thesechanges were significantly dependent upon the natureof interacting proteins; in some cases theirstructures were more ordered. NAD (but not NADH)hampered the formation of streptokinase complexes withdehydrogenases. The plasminogen–activating function ofstreptokinase and the ability of plasminogen to beactivated by streptokinase in the complexes withoxidoreductases were essentially unchanged.Pyruvate kinase induced a moderate (by 35%) increasein the streptokinase activating function. It isassumed that the formation of complexes ofstreptokinase or plasminogen with enzymes may serve asa link in metabolic regulation and/or intercellular interactions. 相似文献
3.
A M Dwivedi R W Woodeshick H L Walton T M Reilly 《Biochemical and biophysical research communications》1991,175(2):437-443
Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form. 相似文献
4.
Incubation of streptokinase in an H2O2-dioxane-bicarbonate buffer (pH 8.5) system leads to the oxidation of tryptophan residues as can be evidenced from the changes in absorption and tryptophan fluorescence spectra. A complete oxidation of tryptophan residues of the protein takes place within 3 hours, the number of the residues is 4. The first tryptophanyl of the protein is oxidized the most easily; the activity of streptokinase decreases thereby by 50%. Modification of the second residue leads to complete inactivation of streptokinase. The rate constants for the oxidation of the first, of the two first and of the third plus fourth tryptophanyls are equal to 1.5.10(-2) min-1, 1,1.10(-2) min-1 and 0.5.10(-2) min -1, respectively. The complete oxidation of tryptophan residues is concomitant with the inability of streptokinase to form stable equimolar complexes with human plasminogen, but in does not result (as can be judged from the CD spectroscopy data) in the breakdown of the protein secondary structure. The specificity of oxidation of the protein tryptophan residues is discussed. The importance of readily oxidized tryptophan residues for the streptokinase function is postulated. 相似文献
5.
The biological function of ??-lactalbumin (??-LA) depends on its conformation. ??-LA can adopt a stable intermediate state induced by heating or pH change. This intermediate state associates with oleic acid (OA) to form an anti-tumor complex. The effect of temperature on the formation the complex has been studied, whereas the effect of pH on complex formation remains unresolved. The effect of pH on tryptophan residues, hydrophobic clusters and secondary structure of Ca2+-depleted bovine ??-LA (BLA) was studied by fluorescence spectroscopy and circular dichroism. BLA was found to adopt a more flexible conformation between pH 7.0 and 9.0 with buried hydrophobic clusters. The binding ability of ??-LA towards OA and the anti-tumor activity of the corresponding complex were also studied. BLA was found to bind more OA over the pH range of 7.0?C9.0 and the corresponding complexes showed a higher anti-tumor activity than those complexes formed under acidic conditions. Our study indicates that alkaline pH aided the formation of the complex as well as its anti-tumor activity. We also propose a possible characteristic structure that facilitates binding of OA. 相似文献
6.
Interactions between bovine γ-globulin (BGG) and borohydride-capped silver nanoparticles (BAgNPs) were studied using dynamic light scattering (DLS) and spectroscopic techniques such as UV–vis spectroscopy, fluorescence, and circular dichroism. The results were compared with earlier reported interactions between γ-globulin and citrate-coated AgNPs (CAgNPs). BAgNPs were synthesized and characterized. Irrespective of the coating on AgNPs, nanoparticles had formed ground-state complexes with the protein. CAgNPs, as well as BAgNPs had caused static quenching of tryptophan (Trp) fluorescence of the protein. The change in the capping agent from citrate to borohydride weakened the binding of nanoparticles with the protein. But the same change in capping agent had increased the fluorescence quenching efficiency of AgNPs. Hydrogen bonding and van der Waals interactions were involved in BGG–BAgNPs complex similar to the CAgNPs complex with γ-globulin. Polarity of the Trp microenvironment in BGG was not altered using BAgNPs as opposed to CAgNPs, as supported using synchronous and three-dimensional fluorescence. Resonance light scattering experiments also suggested nano-bio conjugation. Far-UV and near-UV circular dichroism (CD) spectra respectively pointed towards changes in the secondary and tertiary structure of BGG by BAgNPs, which was not observed for CAgNPs. 相似文献
7.
Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state approximately pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase. 相似文献
8.
Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies 总被引:8,自引:0,他引:8
S A Wharton R W Ruigrok S R Martin J J Skehel P M Bayley W Weis D C Wiley 《The Journal of biological chemistry》1988,263(9):4474-4480
Circular dichroism and tryptophan fluorescence spectroscopy have been used to investigate the structures of the influenza virus membrane glycoprotein hemagglutinin, acid-treated hemagglutinin, and fragments of hemagglutinin derived by proteolysis. The conformational change in hemagglutinin which occurs at the pH of membrane fusion (pH 5-6) was associated with a significant change of the environment of tyrosine residues, a change in the environment of tryptophan residues, but no changes in secondary structure. Tryptic digestion of the hemagglutinin in its low pH conformation which releases one of the subunit polypeptides (HA1) caused minimal changes in tyrosine and tryptophan environments but a small secondary structural change in HA1. The secondary structure of the remainder of the molecule (HA2) was very similar to that predicted from the known x-ray crystallographic structure of the native molecule. However, fluorescence spectroscopy indicated a tertiary change in structure in the coiled coil of alpha-helices which form the fibrous central stem of the molecule. These results are consistent with a conformational change required for membrane fusion which involves a decrease of HA1/HA1, HA1/HA2 interactions and changes in tertiary structure not accompanied by changes in secondary structure. 相似文献
9.
This work studies specific interactions between a small globular protein and a highly flexible, branched polysaccharide using differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, and turbidimetry measurements. It uses the system water/bovine serum albumin (BSA)/dextran (D 2000) as a model. Dextran molecules are able to form interpolymeric complexes with BSA in water at both low and high temperatures if the polysaccharide is in excess and if the protein exists in its associated state. It leads to a partial destabilization of the secondary and tertiary structures of the protein and an additional exposure of the hydrophobic tryptophan residues to the surface of globule. If the total concentration of biopolymers in the mixture is high enough, the stability of the protein molecules with respect to unfolding and thermoaggregation is significantly decreased as a result of an increase in the protein hydrophobicity. 相似文献
10.
Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution 总被引:2,自引:0,他引:2
The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general. 相似文献
11.
Simon M. Cook Amanda Skora Christine M. Gillen Mark J. Walker Jason D. McArthur 《Molecular microbiology》2012,86(5):1052-1062
Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type‐1 and cluster type‐2) with cluster type‐2 being further divided into sub‐clusters type‐2a and type‐2b. In this study, far‐UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type‐2b streptokinase variants could not generate an active site in Glu‐plasminogen through non‐proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type‐2b variants displayed a 29‐ to 35‐fold reduction in affinity for Glu‐plasminogen. All SK variants could activate Glu‐plasminogen when an activator complex was preformed with plasmin; however, type‐2b and type‐1 complexes were inhibited by α2‐antiplasmin. Exchanging skatype‐2a in the M1T1 GAS strain 5448 with skatype‐2b caused a reduction in virulence while exchanging skatype‐2a with skatype‐1 into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism. 相似文献
12.
Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations. 相似文献
13.
Conformational changes induced by binding of ligands to cytosolic NADP(+)-specific isocitrate dehydrogenase from lactating bovine mammary gland were assessed using circular dichroism and fluorescence techniques. The secondary structure of isocitrate dehydrogenase, as monitored by CD spectra in the far-UV region, is unaltered by enzyme-ligand interactions; in contrast, dramatic changes occur in the near-UV region (270-290 nm) assigned to tyrosine and/or solvent-exposed tryptophan residues. Both the coenzyme analog, 2'-phosphoadenosine 5'-diphosphoribose, and NADPH have an effect on the CD spectrum which is opposite to that produced by metal complexes of either isocitrate or citrate. A CD band at 292 nm assigned to approximately 2 tryptophan residues in a hydrophobic environment is unchanged by binding of substrate or coenzyme. Approximately 30% of the intrinsic fluorescence of isocitrate dehydrogenase, corresponding to approximately 2 tryptophan residues, is not quenched by acrylamide in the absence of 6.3 M guanidine hydrochloride and remains unquenched in the enzyme-substrate complex. The constancy in the proportion of buried and exposed tryptophan residues implicates tyrosine in the observed near-UV CD spectral changes. Since binding of ligands does not influence quaternary structure (Seery, V.L., and Farrell, H. M., Jr. (1989) Arch. Biochem. Biophys. 274, 453-462), activation of isocitrate dehydrogenase may be related to a substrate-induced conformational transition. 相似文献
14.
Tatsuo Nehira Kaoru Ishihara Koichi Matsuo Shunsuke Izumi Takeshi Yamazaki Atsuhiko Ishida 《Analytical biochemistry》2012,430(2):179-184
We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca2+ and/or interacting with binding peptides. Because FDCD appears to reflect the protein’s local structure around the fluorophore, it may provide a useful means for “pinpoint analysis” of protein structures. 相似文献
15.
Ward PN Field TR Rosey EL Abu-Median AB Lincoln RA Leigh JA 《Journal of molecular biology》2004,342(4):1101-1114
The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex. 相似文献
16.
R A Copeland H Ji A J Halfpenny R W Williams K C Thompson W K Herber K A Thomas M W Bruner J A Ryan D Marquis-Omer 《Archives of biochemistry and biophysics》1991,289(1):53-61
The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo. 相似文献
17.
Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0-3.5M urea with enzyme inactivation. The compact monomers were characterized by Stokes radius (R(s)) of 40.7A equal to that of globular proteins with the same molecular mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R(s) of 50A, exposed tryptophan residues, and disrupted secondary structure in 8M urea. 相似文献
18.
V N Nikandrov 《The International journal of biochemistry》1992,24(1):47-53
1. Several hypotheses have been advanced to explain the activating function of streptokinase. The predominant hypothesis suggests a stable equimolar streptokinase-plasmin(ogen) complex, activating free plasminogen by an active centre, which is located in the plasmin(ogen) part of the complex. 2. This hypothesis cannot explain a number of phenomena and certain accumulated experimental data, for example: rabbit and bovine plasminogen activation by streptokinase, not forming stable complexes with these plasminogens; possible activation with pH less than or equal to 2, in the presence of urea, during modification of streptokinase tyrosine residues, i.e. when these two proteins cannot form a stable complex. 3. On the basis of acquired experimental data the following concept is suggested: the activating function of streptokinase is oxygen-dependent and is realised with the help of superoxide radical due to the O(2-.)-generating ability of plasminogen and the O(2-.)-converting ability of streptokinase. 相似文献
19.
Mancheño JM De Los Ríos V Martínez Del Pozo A Lanio ME Oñaderra M Gavilanes JG 《Biochimica et biophysica acta》2001,1545(1-2):122-131
Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin. 相似文献
20.
Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy 总被引:1,自引:0,他引:1
Secondary and tertiary structural parameters of two functionally and serologically related proteins, staphylococcal enterotoxins B and C1, have been determined by using circular dichroism and fluorescence spectroscopy. The secondary structures derived from the respective far-UV circular dichroic spectra were 9.5% alpha-helix, 55.0% beta-pleated sheets, 16.5% beta-turns, and 19.0% random coils for enterotoxin B and 15.0% alpha-helix, 38.0% beta-pleated sheets, 25.5% beta-turns, and 21.5% random coils for staphylococcal enterotoxin C1. The values matched well with the secondary structures derived from the amino acid sequences (Chou and Fasman method). Seven antigenic sites have been predicted for both staphylococcal enterotoxins B and C1 by using the hydrophilicity and the secondary structure information. Three of these antigenic sites appear similar. Fluorescence quantum yield of the single tryptophan residue (Trp-197) of both the enterotoxins showed the tryptophan residue in staphylococcal enterotoxin B to be approximately 46% more fluorescent than in staphylococcal enterotoxin C1. Tryptophan fluorescence quenching by the surface quencher I- and the neutral quencher acrylamide revealed that the single tryptophan residue in each of the enterotoxins is buried in the protein matrix and is not accessible to the surface quencher I-. The tryptophan residue in staphylococcal enterotoxin C1 is 14% less accessible to acrylamide than in staphylococcal enterotoxin B. The data, in general, reflect several similarities and significant differences between the two related enterotoxins. 相似文献