共查询到20条相似文献,搜索用时 15 毫秒
1.
Bugs MR Bortoleto-Bugs RK Cornélio ML Ward RJ 《Biochemical and biophysical research communications》2007,353(4):889-894
We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant. 相似文献
2.
Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca(2+)-independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca(2+)-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca(2+)-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. 相似文献
3.
Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge. 相似文献
4.
Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis. 总被引:1,自引:0,他引:1
C P Dietrich J F Paiva R A Castro S F Chavante W Jeske J Fareed P A Gorin A Mendes H B Nader 《Biochimica et biophysica acta》1999,1428(2-3):273-283
A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins. 相似文献
5.
Kinetic analysis of various heparin fractions and heparin substitutes in the thrombin inhibition reaction 总被引:1,自引:0,他引:1
Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction. 相似文献
6.
The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin. 相似文献
7.
Heparin is a mixture of linear polysaccharides of undetermined sequence. Both biosynthetic data and computer simulation studies have established that each heparin polymer chain is comprised of oligosaccharides of defined sequence, representing ordered domains. One such ordered domian is a pentasaccharide corresponding to heparin's antithrombin III binding site. Previous computer simulation studies, performed under the assumption that heparin lyase (heparinase, EC 4.2.2.7), has a random endolytic action pattern, suggested that certain of these ordered oligosaccharide domains may themselves be nonrandomly arranged in the heparin polymer. The present work presents computer simulations of alternative action patterns for heparin lyase while assuming a random distribution of these oligosaccharide units within the heparin polymer. We consider action patterns that are determined solely by the primary structure of the substrate molecules. Results of the simulations are compared to (1) the experimental measurements of product chains formed throughout the reaction and (2) the change in weight average molecular weight Mw as a function of reaction completion as determined by absorbance at 232 nm. From the simulation of 60 action patterns for heparin lyase, we infer that one of the following statements concerning heparin and heparin lyase is true: (1) Heparin is a random arrangement of a small number of structurally defined oligosaccharide units. Heparin lyase changes its action pattern during the depolymerization of heparin (perhaps influenced by the secondary structure of substrate). (2) Heparin contain clusters of oligosaccharide sequences that are present in low concentrations (overall) in the polymer. Heparin lyase has a specificity for cleaving glycosidic linkages either exolytically at the nonreducing terminus of a chain or (endolytically) at the reducing side of these rare oligosaccharide sequence. 相似文献
8.
R J Linhardt H M Wang D Loganathan J H Bae 《The Journal of biological chemistry》1992,267(4):2380-2387
The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins. 相似文献
9.
Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Multiple complexes of thrombin and heparin 总被引:2,自引:0,他引:2
Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin. 相似文献
11.
S T Olson I Bj?rk R Sheffer P A Craig J D Shore J Choay 《The Journal of biological chemistry》1992,267(18):12528-12538
The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
Bannikova GE Stolbushkina PP Drozd NN Vikhoreva GA Varlamov VP Makarov VA Panov AV 《Prikladnaia biokhimiia i mikrobiologiia》2004,40(4):429-434
The possibility of obtaining low-molecular-weight heparins using the chitinolytic enzymatic complex immobilized on Silochrom has been demonstrated. The optimal conditions of this process (sodium acetate buffer, pH 7.0-7.5; temperature, 40-45 degrees C; and duration of hydrolysis, 3 h) were determined. Depending on the ratio between heparin and the immobilized enzymatic complex, samples with molecular weight varying from 1.7 to 4.7 kDa, were obtained. These complexes inhibited factor Xa in 2.0-3.7 times more effectively than original heparin. 相似文献
13.
A D Cardin R L Jackson B Elledge D Feldhake 《International journal of biological macromolecules》1989,11(1):59-62
Successive rechromatography of commercial bovine lung heparin on human plasma low density lipoproteins (LDL) immobilized to AffiGel-10 yielded four high reactive heparin (HRH-I to IV) fractions and an unreactive fraction (URH). HRH-I was the most sulphated HRH fraction whereas URH had the least sulphation. In the presence of 10 mM Ca2+, LDL were precipitated by these heparins in the following order: HRH-II greater than HRH-III greater than HRH-IV greater than HRH-I greater than URH. The average molecular weight of HRH-I to IV was 8600, 11400, 10,100, and 10,000, respectively. A plot of log molecular weight versus the concentration of HRH required to give half-maximal precipitation of LDL showed a negative correlation (r = -0.880). These results indicate that heparin chain length is an important determinant of heparin binding to LDL in solution and may have relevance to the binding and precipitation of LDL in the arterial wall. 相似文献
14.
A L Stone 《Archives of biochemistry and biophysics》1985,236(1):342-353
The therapeutic anticoagulant action of heparin is mediated by the ability of a multifunctional octadecasaccharide region of the molecule to bind to and differentially alter the conformational integrity of antithrombin, and the sugar sequence of the primary binding domain is known. Low ultraviolet circular dichroism spectroscopy of heparin-derived anticoagulant octa-, deca-, dodeca-, tetradeca-, and octadecasaccharides has been useful in elucidating the nature of the sugars that are contained within the second functional domain of the octadecasaccharide. The difference between the spectra of the molar ellipticity of the above sequential oligosaccharides was taken to be the CD spectrum of the corresponding additional disaccharide unit(s). Optical models of the component disaccharides of heparin were derived from CD spectra of heparins having a high degree of sulfation, synthetic glycosides of N-acetylglucosamine, and glycosides of component uronic acids. These were sufficiently distinct in magnitude and spectral position to warrant interpretation of the experimental difference CD spectra. The uronic acids of the disaccharides between deca- and octamer, dodeca- and decamer, and tetradeca- and dodecamer were thereby ascribed to sulfated iduronate, unsulfated iduronate, and glucuronate residues, respectively, while those of the tetrasaccharide between the octadeca- and tetradecasaccharide were tentatively assigned to sulfated iduronate moieties. Interpretation of the difference CD spectra on the basis of the optical models was less certain in regard to the amino sugar components. It appears that the amino sugar derivative between the dodeca- and decamer was N-acetylglucosamine, while the other disaccharides of the octa- to octadecasaccharide probably contained the N-sulfated derivative. A speculative disaccharide sequence drawn from these data indicates that relatively less strongly anionic disaccharides, having nonsulfated uronic acid moieties and N-acetylglucosamine, were flanked by trisulfated disaccharide units, constituting a structural element similar to that which contains the primary binding domain of the anticoagulant. 相似文献
15.
The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters. 相似文献
16.
The role of surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of alpha-thrombin 总被引:3,自引:0,他引:3
C C Heuck U Schiele D Horn D Fronda E Ritz 《The Journal of biological chemistry》1985,260(8):4598-4603
We have compared surface charge and the surface charge density on the polyanions heparin and potassium polyvinyl sulfate (KPVS), as well as on hydrolyzed heparin and KPVS, with their accelerating effect on the inhibitory action of antithrombin III on thrombin. Polyelectrolyte titration of thrombin with KPVS or heparin at pH 7.4 clearly indicates an electrostatic interaction. In contrast, at the same pH no electrostatic interaction is observed between polyanions and antithrombin III. KPVS accelerates the inhibitory action of antithrombin III to the same extent as heparin on the basis of charge equivalence. Heparin and KPVS with a mean distance between two charged centers of less than 0.75 and 0.95 nm, respectively, accelerate strongly whereas hydrolysates with lower charge densities are far less active. The following observations are indicated. Intramolecular neutralization of oppositely charged residues occurs within thrombin, antithrombin III, and partially hydrolyzed heparin. Heparin acts on the antithrombin III-thrombin reaction through cooperative electrostatic binding to thrombin and nonelectrostatic interaction with antithrombin III. This indicates a quasi-catalytic action of the polyelectrolyte. Hydrolysis of only a few N-sulfate residues within the heparin molecule decreases the linear surface charge density to such an extent that the accelerating action is drastically reduced. The loss of accelerating capacity agrees with the sudden loss of counterion condensation due to the decrease of the linear surface charge density beyond limits postulated by Manning in a theory of polyelectrolytes. 相似文献
17.
In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N- and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N- or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity. 相似文献
18.
A. Leizorovicz M. C. Haugh F. R. Chapuis M. M. Samama J. P. Boissel 《BMJ (Clinical research ed.)》1992,305(6859):913
OBJECTIVE--To determine whether prophylactic treatment with low molecular weight heparin reduces the incidence of thrombosis in patients who have had general or orthopaedic surgery. DESIGN--Meta-analysis of results from 52 randomised, controlled clinical studies (29 in general surgery and 23 in orthopaedic surgery) in which low molecular weight heparin was compared with placebo, dextran, or unfractionated heparin. SUBJECTS--Patients who had had general or orthopaedic surgery. INTERVENTION--Once daily injection of a low molecular weight heparin compared with placebo, dextran, or unfractionated heparin. MAIN OUTCOME MEASURES--Incidence of deep venous thrombosis, pulmonary embolism, major haemorrhages, and death. RESULTS--The results confirm that low molecular weight heparins are more efficacious for the prophylactic treatment of deep venous thrombosis than placebo (common odds ratio 0.31, 95% confidence interval 0.22 to 0.43; p < 0.001) and dextran (0.44, 0.30 to 0.65; p < 0.001). The results suggest that low molecular weight heparins are also more efficacious than unfractionated heparin (0.85, 0.74 to 0.97; p = 0.02), with no significant difference in the incidence of major haemorrhages (1.06, 0.93 to 1.20; p = 0.62). CONCLUSIONS--Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. However, it remains to be shown in a suitably powered clinical trial whether low molecular weight heparin reduces the risk of fatal pulmonary embolism compared with heparin. 相似文献
19.
Marc Torrent M. Victòria Nogués Ester Boix 《Journal of molecular recognition : JMR》2011,24(1):90-100
The eosinophil cationic protein (ECP) is an eosinophil‐secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. During inflammation and eosinophilia disorders, ECP is secreted to the inflammation area, where it would contribute to the immune response. ECP secretion causes also severe damage to the host own tissues. ECP presents a high affinity for heparin and this property might be crucial for its immunomodulating properties, antipathogen action, and its toxicity against eukaryotic cells. ECP, also known as human RNase 3, belongs to the mammalian RNase A superfamily and its RNase activity is required for some of its biological properties. We have now proven that ECP heparin binding affinity depends on its RNase catalytic site, as the enzymatic activity is blocked by heparin. We have applied molecular modeling to analyze ECP binding to heparin representative probes, and identified protein residues at the catalytic and substrate binding sites that could contribute to the interaction. ECP affinity for heparin and other negatively charged glycosaminoglycans (GAGs) can explain not only its binding to the eukaryote cells glycocalix but also the reported high affinity for the specific carbohydrates at bacteria cell wall, promoting its antimicrobial action. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
20.
Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments 总被引:1,自引:0,他引:1
P Schoen T Lindhout G Willems H C Hemker 《The Journal of biological chemistry》1989,264(17):10002-10007
Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin. 相似文献