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1.
Hyaluronan (HA) is the substrate of hyaluronidase (HAase). In addition, HA is able to form electrostatic complexes with many proteins, including HAase. Experiments have shown the strong inhibition of the HA hydrolysis catalyzed by HAase when performed at low HAase over HA concentration ratio and under low ionic strength conditions. Non-catalytic P proteins are able to compete with HAase to form electrostatic complexes with HA and thus to modulate HAase activity. We have modeled the HA–HAase–P system by considering the competition between the two complex equilibria HA–P and HA–HAase, the Michaelis–Menten type behavior of HAase, and the non-activity of the electrostatically complexed HAase. Simulations performed by introducing experimental data produce a theoretical behavior similar to the experimental one, including all the atypical phenomena observed: substrate-dependence, enzyme-dependence and protein-dependence of HAase. This shows that our assumptions are sufficient to explain the behavior of the system and allow us to estimate unknown parameters and suggest new developments.  相似文献   

2.
Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.  相似文献   

3.
Hyaluronan (HA) hydrolysis catalyzed by hyaluronidase (HAase) is inhibited at low HAase over HA ratio and low ionic strength, because HA forms electrostatic complexes with HAase, which is unable to catalyze hydrolysis. Bovine serum albumin (BSA) was used as a model to study the HA-protein electrostatic complexes at pH 4. At low ionic strength, there is formation of (i) neutral insoluble complexes at the phase separation and (ii) small positively-charged or large negatively-charged soluble complexes whether BSA or HA is in excess. According to the ionic strength, different types of complex are formed. Assays for HA and BSA led to the determination of the stoichiometry of these complexes. HAase was also shown to form the various types of complex with HA at low ionic strength. Finally, we showed that at 0 and 150 mmol L(-1) NaCl, BSA competes with HAase in forming complexes with HA and thus induces HAase release resulting in a large increase in the hydrolysis rate. These results, in addition to data in the literature, show that HA-protein complexes, which can exist under numerous and varied conditions of pH, ionic strength and protein over HA ratio, might control the in vivo HAase activity.  相似文献   

4.
Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biological properties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst the substrate-dependence curve has a Michaelis-Menten shape with a 0.15 mol L(-1) ionic strength, at low ionic strength (5 mmol L(-1)), a strong decrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximum increases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by the ability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify this hypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albumin then was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to be free and catalytically active. The kinetic results showed that the HA-HAase non-specific complex inhibits HAase catalytic activity towards HA. Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induce another type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition or activation, in particular under in vivo conditions, is discussed.  相似文献   

5.
A dual‐signal strategy is proposed based on fluorescent biomass‐based carbon dots (BC‐dots) and chitosan stabilized AuNPs (CS@AuNPs) to determine hyaluronidase (HAase). BC‐dots can induce aggregation of CS@AuNPs nanoparticles with a colour change from red to blue. Positively charged CS@AuNPs interacted with the negatively charged hyaluronic acid (HA) through electrostatic adsorption, and CS@AuNPs maintained stability due to the semirigid coil conformation of HA. However, in the presence of HAase, due to enzymatic hydrolysis of HA by HAase, the CS@AuNPs agglomerated. Based on the change of fluorescence and colour, quantitative analysis of HAase was achieved. Linear ranges for the fluorometric and colorimetric determinations were 2.0–70 U mL?1 and 8–60 U mL?1, respectively, with a detection limit of 0.27 U mL?1. This dual‐signal sensing system possesses high potential for determination of HAase in biological matrices.  相似文献   

6.
Botulinum neurotoxin is produced by Clostridium botulinum and forms large protein complexes through associations with nontoxic components. We recently found that hemagglutinin (HA), one of the nontoxic components, disrupts the intercellular epithelial barrier; however, the mechanism underlying this phenomenon is not known. In this study, we identified epithelial cadherin (E-cadherin) as a target molecule for HA. HA directly binds E-cadherin and disrupts E-cadherin–mediated cell to cell adhesion. Although HA binds human, bovine, and mouse E-cadherin, it does not bind rat or chicken E-cadherin homologues. HA does not interact with other members of the classical cadherin family such as neural and vascular endothelial cadherin. Expression of rat E-cadherin but not mouse rescues Madin–Darby canine kidney cells from HA-induced tight junction (TJ) disruptions. These data demonstrate that botulinum HA directly binds E-cadherin and disrupts E-cadherin–mediated cell to cell adhesion in a species-specific manner and that the HA–E-cadherin interaction is essential for the disruption of TJ function.  相似文献   

7.
8.
Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.  相似文献   

9.
Hyaluronan (HA) has different biological functions according to its molar mass; short HA fragments are involved in inflammation processes and angiogenesis, whereas native HA is not. Physicochemically, studies of native HA hydrolysis catalyzed by bovine testicular hyaluronidase (HAase) have suggested that kinetic parameters depend on HA chain length. To study the influence of HA chain length in more detail, and to try to correlate the physicochemical and biological properties of HA, HA hydrolysis catalyzed by HAase was used in a new procedure to obtain HA fragments of different molar masses. HA fragments (10-mg scale) with a molar mass from 800 to 300,000 g mol(-1) were prepared, purified using low-pressure size exclusion chromatography (SEC), lyophilized, and characterized in molar mass by either mass spectrometry or HPLC-SEC-multiangle laser light scattering. The polydispersity index of the purified fractions was less than 1.25. The complete set of HA standards obtained was used to calibrate our routine HPLC-SEC device using only a refractive index (RI) detector. We showed that the N-acetyl-d-glucosamine reducing end assay and the calibrated HPLC-SEC-RI gave equivalent kinetic data. In addition, the HPLC-SEC-RI furnished the mass distribution of the polysaccharide during its hydrolysis.  相似文献   

10.
The light scattering of bovine serum albumin (BSA) has been measured at protein concentration up to 90 g/L and at pH values between 4.4 and 7.6. The dependence of scattering on both protein concentration and pH may be quantitatively accounted for by a simple extension of the hard-sphere model for protein solutions [Ross, P. D. & Minton, A. P. (1977) J. Mol. Biol. 112 , 437–452] allowing for electrostatic repulsions between molecules. According to the extended model, the radius of the effective hard spherical particle representing BSA varies with the net electrical charge of the BSA molecule in a manner which may be calculated from electrostatic theory.  相似文献   

11.
The atomic and electronic structures of heme complexes with His, Gly, and Cys residues (Heme–His, Heme–Gly, and Heme–Cys) in the fifth coordination position of the Fe atom and with oxygen and nitrogen oxide molecules in the sixth Fe position were studied by the semiempirical quantum-chemical method PM3. A comparative analysis of internuclear distances showed that the strength of chemical bonding between the ligand molecules (oxygen and nitrogen oxide) is greater for Heme–Cys than for Heme–His and Heme–Gly complexes. Consequently, the strengthening of the chemical bond of the oxygen (or nitrogen oxide) molecule with Heme–Cys substantially weakens the chemical bond in the ligand molecule. The Mulliken population analysis showed that the electronic density of ligand (oxygen or nitrogen oxide) p-orbitals is transferred to the d-orbitals of the Fe atom, whose charge, calculated according to the Mulliken analysis, formally becomes negative. In the Heme–His complex with oxygen, this charge is substantially greater than in the complex with NO, and the oxygen molecule becomes polarized. No oxygen polarization is observed in the Heme–Cys complex, and the electron density (judging from the change in the Fe charge) is transferred to the coordinated sulfur atom. This is also characteristic of Heme–Cys complexes with nitrogen oxide. An analysis of charges on the atoms indicates that the character of chemical bonding of the oxygen molecule in Heme–Cys and Heme–Gly complexes is similar and basically differs from that in the case of the Heme–His complex.  相似文献   

12.
Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA– calnexin–calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.  相似文献   

13.
Hyaluronan (HA) oligosaccharides which can stimulate angiogenesis and suppress the growth of tumors have attracted more and more attention. In order to prepare pure and well-defined oligosaccharides from high-molecular-weight HA in a rapid and simple manner, an enzymatic degradation method was developed, which included degradation with a novel recombinant hyaluronan lyase (HA lyase, hyaluronidase, or HAase) and gel permeation chromatography. The HAase protein was expressed in Escherichia coli with the expression vector pBV220. The HAase was purified and refolded, and specific activity of the enzyme solution was 3800 U/mg. HA was degraded with HAase at the optimized conditions, yielding 46% and 31% of HA disaccharides and HA tetrasaccharides, respectively. These HA oligosaccharides were conveniently separated by consecutive column chromatography on Bio-gel P6 and were identified by HPLC–MS.  相似文献   

14.
Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.  相似文献   

15.
Hyaluronic acid (HA) is a polysaccharide widely used in biomedical applications, due to its elevated biocompatibility and the peculiar viscoelastic properties of its solutions. Although the viscoelastic behaviour of HA solutions has been extensively studied in the literature it has been often reported in the range of low frequency (1-100 Hz) and high salt concentration, whereas the main rheological peculiarities of this molecule are expected at high frequency (>100 Hz) and low salt concentration. In this work we studied the viscoelastic properties of low molecular weight HA (155 kDa) in wide range of concentrations (0.01-20 mg/ml) at low ionic strength and over an extended frequency range (0.1-1000 Hz) using both optical tweezers and conventional rheometry. Good agreement between the high frequency dynamic behaviour (optical tweezers) and the viscoelastic properties at low frequency (rheometry) was found. We also found that, in apparent contradiction with polyelectrolyte solution theory, HA solution behaves as liquid-like viscoelastic fluid (G'>G') even at concentrations higher than the entanglement concentration where a weak-gel behavior should be expected.  相似文献   

16.
The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.  相似文献   

17.
This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10–5(per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 104M–1(per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20°C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.  相似文献   

18.
As a contribution to the understanding of the thermodynamic state of single salts in living systems, the activity coefficients of KCl were determined in concentrated bovine serum albumin (BSA) solutions. The concentration range studied was 0.01 to 0.5 M KCl and zero to 18% wt BSA, thus amply covering physiological conditions. The activity coefficients of the salt were measured using the EMF method with ion exchange membrane electrodes. Keeping the salt concentration constant, the activity coefficients of the salt decrease linearly with protein concentration, the effect being more pronounced for low salt content. The maximal deviations of the activity coefficients with respect to those in pure salt solution amount to ca. 40% for 0.01 M KCl and 18% wt BSA. The results were interpreted on the assumption of the superposition of three effects i.e. water bound to BSA molecules as non-solvent water, specific Cl ion binding and the electrostatic interactions of the polyions with the salt ions. In view of the results it can be concluded that only a small portion of simple intracellular ions are bound, based on the assumption that the cytoplasm of living cells may be regarded as a concentrated protein-salt solution.  相似文献   

19.
The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than ±1. 5 μm s(-1) V(-1) cm(-1). Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ~60 BSA molecules for every GA molecule, while at pH 5 there were ~10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.  相似文献   

20.
Hyaluronidase (HAase), a class of enzymes which degrade hyaluronic acid (HA), are involved in the spread of infections/toxins, ovum fertilization, and cancer progression. Thus, HAase inhibitors may have use in disease treatments. We evaluated 21 HAase inhibitors against HYAL-1, testicular, honeybee, and Streptomyces HAases. Among these inhibitors, polymers of poly (styrene-4-sulfonate) (PSS) (i.e., molecular weight 1400-990,000 or PSS 1400-PSS 990,000) and O-sulfated HA (sHA) derivatives (sHA2.0, 2.5, and 2.75) were the most effective. HYAL-1 and bee HAases were the most sensitive, followed by testicular HAase; Streptomyces HAase was resistant to all inhibitors, except PSS 990,000 and VERSA-TL 502 (i.e., PSS 10(6) dalton). The length of the PSS polymer determined their potency (e.g., IC50 for HYAL-1, PSS 990,000: 0.0096 microM; PSS 210 no inhibition; IC50 for testicular HAase, PSS 990,000: 0.042 microM; PSS 210 no inhibition). The presence, but not the number, of sulfate groups on the sHA molecule determined its potency (e.g., IC50 for HYAL-1: sHA2.0, 0.019 microM; sHA2.75, 0.0083 microM). Other known HAase inhibitors, such as gossypol, sodium-aurothiomalate, 1-tetradecane sulfonic acid, and glycerrhizic acid, were not effective. Both PSS and sHA inhibited HAases by a mixed inhibition mechanism (i.e., competitive + uncompetitive) and were 5- to 17-fold better as uncompetitive inhibitors than as competitive inhibitors. These results demonstrate that HAase inhibitors show selectivity toward the different types of HAases, which could be exploited to inhibit specific HAases involved in a variety of pathophysiologic conditions.  相似文献   

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