共查询到20条相似文献,搜索用时 15 毫秒
1.
Some structural properties of Neurospora tyrosinase have been studied by fluorescence spectroscopy. The emission spectra observed for oxy-, deoxy-, met- and apo-tyrosinase and the Co2+-substituted form are indicative of a protein containing buried tryptophan residues. By using acrylamide and iodide, part of the emission is quenched, indicating heterogeneity in the tryptophan environment. Upon binding of Cu2+ or Co2+ to apo-tyrosinase, a marked decrease of the tryptophan quantum yield is observed. A further decrease in emission intensity results from the binding of molecular O2 to the deoxy form. The fluorescent probe 8-anilinonaphthalene-1-sulphonate binds to tyrosinase only when the metal ions are removed. Reconstitution of apo-tyrosinase with Cu2+ completely displaces the probe, suggesting that 8-anilinonaphthalene-1-sulphonate binds to apo-tyrosinase at the active site. The fluorescence properties of Neurospora tyrosinase are compared with those of haemocyanin. 相似文献
2.
The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides. 相似文献
3.
Proteolytic formation and properties of a fragment of protein C containing the gamma-carboxyglutamic acid rich domain and the EGF-like region 总被引:1,自引:0,他引:1
The function of the epidermal growth factor (EGF) like domains in the vitamin K dependent plasma proteins is largely unknown. In order to elucidate the function of these domains in protein C, we have devised a method to isolate the EGF-like region from the light chain connected to the NH2-terminal region, containing the gamma-carboxyglutamic acid (Gla) residues. This was accomplished by tryptic cleavage of protein C that had been reversibly modified with citraconic anhydride to prevent cleavage at the lysine residue (in position 43) that is located between the two regions. The isolated fragment consists of residues 1-143 from the light chain of protein C connected by a disulfide bond to residues 108-131 from the heavy chain. Upon Ca2+ binding to the isolated Gla-EGF fragment from bovine protein C, the tryptophan fluorescence emission was quenched in a manner indicating binding to at least two classes of binding sites. These were presumably the Gla-independent Ca2(+)-binding site located in the EGF-like region and the lower affinity sites in the Gla region. A comparison with the tryptophan fluorescence quenching that occurred upon Ca2+ binding to the separately isolated EGF-like and Gla regions suggested that the EGF-like region influenced the structure and Ca2+ binding of the Gla region. The isolated Gla-EGF fragment functioned as an inhibitor of the anticoagulant effect of activated protein C in a clotting assay, whereas no inhibition was observed with either the Gla region or the EGF-like region. 相似文献
4.
Interaction of alpha-lactalbumin with Cu2+ 总被引:1,自引:0,他引:1
It has been shown by intrinsic fluorescence spectroscopy that alpha-lactalbumin has several Cu2+ -binding sites per molecule. The Ca2+ -loaded protein binds two or more Cu2+ per molecule with an association constant of about 3 X 10(3) M-1. Apo-alpha-lactalbumin binds one Cu2+ per molecule with association constant 8 X 10(4) M-1 and from two to three Cu2+ with an association constant of about 4 X 10(3) M-1. The results obtained from spectrofluorometric pH titration of alpha-lactalbumin in the acidic pH region show the possible involvement of histidine residues in the coordination of Cu2+. The binding of Cu2+ to alpha-lactalbumin lowers significantly its thermostability and stability towards urea denaturation. The stability of Cu2+, Ca2+-alpha-lactalbumin against thermal and urea denaturation is similar to that of the apo protein. The thermal transition in Cu2+, Ca2+-alpha-lactalbumin occurs within the region of physiological temperatures which may suggest the existence of some thermal regulation of its functioning in vivo. 相似文献
5.
Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character. 相似文献
6.
Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+ 相似文献
7.
Wells MA Jackson GS Jones S Hosszu LL Craven CJ Clarke AR Collinge J Waltho JP 《The Biochemical journal》2006,399(3):435-444
It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo. 相似文献
8.
J R Pearlstone T Borgford M Chandra K Oikawa C M Kay O Herzberg J Moult A Herklotz F C Reinach L B Smillie 《Biochemistry》1992,31(28):6545-6553
A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
Interaction of fatty acids with the calcium-magnesium ion dependent adenosinetriphosphatase from sarcoplasmic reticulum 总被引:1,自引:0,他引:1
The fluorescence emission spectrum of dansylundecanoic acid is sensitive to the environment and appears at a lower wavelength when the fatty acid is bound to protein than when it is bound to phospholipid. When bound to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum, the emission spectrum can be resolved into separate components assigned to fatty acid bound to protein and to lipid. Efficiency of energy transfer from the tryptophan residues of the ATPase to dansylundecanoic is higher for protein-bound probe than for lipid-bound probe. Fluorescence titrations are consistent with three fatty acid binding sites per ATPase with a Kd of 7 microM, and these sites are postulated to occur at the protein-protein interface in ATPase oligomers. Fatty acid incorporated into the lipid component of the membrane appears to be bound outside the lipid annulus around the protein. 相似文献
10.
Preferred heme binding sites of histidine-rich glycoprotein 总被引:1,自引:0,他引:1
The heme binding sites of rabbit histidine-rich glycoprotein (HRG), 94 kDa, were studied with rose bengal (RB), a fluorescein derivative that associates with histidine residues. Difference absorbance spectra indicate that HRG binds RB at two thermodynamically preferred sites (Kd approximately 2 microM) that are spectroscopically equivalent. Up to 18-22 equiv of RB can also be bound by a set of lower affinity sites. Mesoheme is capable of displacing RB from the two preferred sites (Kd = 0.6 microM) and provides evidence that the two sites are not identical. Two peptides isolated from plasmin-digested HRG, one 35-kDa peptide rich in histidine (approximately 30 mol %) and one 15-kDa peptide relatively poor in histidine (approximately 4 mol %), also bind RB and mesoheme. The two preferred RB binding sites of HRG are located on the 15-kDa histidine-poor peptide and the lower affinity "class" of sites on the 35-kDa histidine-rich peptide. Mesoheme or RB quenches the tryptophan fluorescence of HRG and the histidine-poor peptide with an apparent binding stoichiometry near 2. Fluorescence quenching also indicates that 1-2 equiv of Cu(II) binds to the 15-kDa peptide, and absorbance spectroscopy provides evidence that Cu(II) is capable of displacing heme from the peptide. The fluorescence lifetimes of RB, determined by phase-modulation fluorometry, indicate that the two preferred sites in the histidine-poor domain are more apolar than the more numerous sites located in the histidine-rich region of the protein. 相似文献
11.
We report the biochemical characterization of calhepatin, a calcium-binding protein of the S100 family, isolated from lungfish (Lepidosiren paradoxa) liver. The primary structure, determined by Edman degradation and MS/MS, shows that the sequence identities with the other members of the family are lower than those between S100 proteins from different species. Calhepatin is composed of 75 residues and has a molecular mass of 8670 Da. It is smaller than calbindin D(9k) (78 residues), the smallest S100 described so far. Sequence analysis and molecular modelling predict the two EF-hand motifs characteristic of the S100 family. Metal-binding properties were studied by a direct 45Ca2+-binding assay and by fluorescence titration. Calhepatin binds Ca2+ and Cu2+ but not Zn2+. Cu2+ binding does not change the affinity of calhepatin for Ca2+. Calhepatin undergoes a conformational change upon Ca2+ binding as shown by the increase in its intrinsic fluorescence intensity and lambda(max), the decrease in the apo-calhepatin hydrodynamic volume, and the Ca2+-dependent binding of the protein to phenyl-Superose. Like most S100 proteins, calhepatin tends to form noncovalently associated dimers. These data suggest that calhepatin is probably involved in Ca2+-signal transduction. 相似文献
12.
Terbium ions bind with a 2:1 stoichiometry per subunit to inorganic pyrophosphatase from bakers' yeast (EC 3.6.1.1) as measured by an increase of terbium fluorescence. The Tb3+ inhibition of the Mg2+ activated pyrophosphate hydrolysis is caused by a competitive binding at the substrate site of the active centre. The second Mg2+ binding site--the so-called "stabilization site"--is discussed as an additional binding site for Tb3+. Thereby, Tb3+ causes also a stabilization of the enzyme against heat denaturation. The dissociation constants of the terbium-pyrophosphatase interaction are in the micromolar range. 相似文献
13.
Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus
A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue. 相似文献
14.
Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7. 相似文献
15.
E A Permiakov L A Kalinichenko L A Morozova Iu V Derezhkov J Bagelova 《Molekuliarnaia biologiia》1988,22(4):984-991
Interactions of the calcium binding proteins, parvalbumin from cod muscles, alpha-lactalbumin from cow milk and calmodulin from bovine brain, with Cu2+ and Zn2+ ions have been studied by intrinsic fluorescence and microcalorimetry methods. It was revealed that parvalbumin binds one Cu2+ ion per molecule with association constant from 10(5) to 10(6) M-1. Zn2+ ions seem to compete for the same site which does not coincide with the two Ca2+ and Mg2+ binding sites. alpha-Lactalbumin contains from 2 to 4 Cu2+ and Zn2+ binding sites, the number and affinities of which depend on Ca2+ concentration. Calmodulin has similar Cu2+ and Zn2+ binding sites. The binding of Cu2+ and Zn2+ ions to parvalbumin and alpha-lactalbumin changes the shape and position of their thermal denaturation transitions. The results obtained together with the literature data show that the ability to interact with Cu2+ and Zn2+ ions is a property inherent to many calcium-binding proteins, which may play a physiological role for some of them. 相似文献
16.
Jones CE Klewpatinond M Abdelraheim SR Brown DR Viles JH 《Journal of molecular biology》2005,346(5):1393-1407
The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease. 相似文献
17.
We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding. 相似文献
18.
The fluorescence titration curve of skeletal muscle troponin containing TnI with 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and/or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+ binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. 相似文献
19.
Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that the pH has a marked effect on the fluorescence intensity of holo-ACF II; however, no appreciable shift of the emission maximum of holo-ACF II was observed in the pH range of 3-10. It was deduced from a relatively weak fluorescence emission of holo-ACF II at a neutral pH (6-7) that native holo-ACF II assumes a compactly folded structure in which the most interior Trp residues and quenchers are adjacent. Terbium ions can completely replace both Ca2+ ions in holo-ACF II as determined by equilibrium dialysis. Two Tb3+-binding sites with different apparent Tb3+ association constant values, (2.1 +/- 0.2) and (1.0 +/- 0.1) x 10(7) M(-1), were identified through Tb3+ fluorescence titration. In addition, it was confirmed from the titration of holo-ACF II and Tb3+-ACF II with N-bromosuccinimide (NBS) that only interior Trp residues are involved in the energy transfer to Tb3+ ions and all accessible Trp residues located in the surface of holo-ACF II have a similar affinity to NBS while those located in the surface of Tb3+-ACF II have two different kinds of affinity to NBS, which suggests a conformational change of holo-ACF II on the substitution of Tb3+ for Ca2+. 相似文献
20.
Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein 总被引:1,自引:0,他引:1
The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it. 相似文献