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1.
Martin B. Mathews LaVerne Decker 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,498(1):259-263
Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules. 相似文献
2.
L Rosenberg L H Tang S Pal T L Johnson H U Choi 《The Journal of biological chemistry》1988,263(34):18071-18077
When link protein binds to hyaluronate in the absence of proteoglycan monomer a high molecular weight complex is formed. Two assay procedures have been developed to examine the formation of the complex and the rate and stoichiometry of binding of link protein to hyaluronate in the complex. In the first, the complex is isolated by differential centrifugation, and the stoichiometry of binding of link protein to hyaluronate in the sedimented complex is determined. In the second assay, which involves turbidimetry, the rate of complex formation (delta A420/min) is determined, and the amount of complex formed is determined in terms of the maximum turbidity (A420,max) attained. The effects of temperature, pH, initial total solute concentration, and the ratio by weight of link protein to hyaluronate on the amount of complex formed and on the rate of complex formation were examined. There is a linear correlation between the amount of complex formed as determined by turbidity and by differential centrifugation. Using these assays, we examined the specificity of the binding of link protein to hyaluronate and the capacity of hyaluronate oligosaccharides to competitively inhibit the binding of link protein to hyaluronate. Hyaluronate decasaccharide is the oligosaccharide of minimum size that strongly inhibits the binding of link protein to hyaluronate. Proteoglycan monomers dissociate from hyaluronate as the pH is decreased from pH 7 to pH 5. Turbidimetric studies show that the rate of binding of link protein to hyaluronate increases with decreasing pH. The binding affinity of proteoglycan monomers for hyaluronate is decreased at pH 5, whereas the binding affinity of link protein for hyaluronate is not. This difference in the effect of pH on the stability of binding of link protein to hyaluronate, compared with proteoglycan monomer, explains in part the capacity of link protein to stabilize the binding of proteoglycan monomer to hyaluronate at pH 5. 相似文献
3.
Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells 总被引:7,自引:0,他引:7
The binding of labeled hyaluronate to the surface of Simian virus 40-transformed 3T3 cells was studied as a function of 1) pH, 2) ionic strength, 3) temperature, and 4) molecular weight of the hyaluronate. Binding occurred over a wide range of pH values with optima at pH 7 and at less than pH 4. Binding at low pH was eliminated at high ionic strength whereas that at physiological pH was enhanced, with a maximum at 0.5 M NaCl. The enhancement of binding at pH 7 was reversible and independent of the particular salt used. Scatchard plot analysis showed that increasing the ionic strength resulted in both a decrease in the dissociation constant (Kd) and an increase in the amount bound at saturation (Bmax). Temperature also influenced the binding of hyaluronate to the cell surface. The amount bound at low temperatures (0 degrees C) was 3 to 5 times that bound at high temperatures (40 degrees C) with a sharp transition occurring at 18 degrees C, the temperature of phase transition of the plasma membrane. The temperature effect was primarily a change in the Bmax and was reversible. Finally the molecular weight of the ligand influenced the binding. High molecular weight preparations of hyaluronate had a higher binding affinity (lower Kd) and a lower Bmax than did smaller molecular weight preparations. 相似文献
4.
The study of the Cu(II)-hyaluronate complexes by absorption and CD spectra, as well as by acid–base titration and viscosity, provides information about the nature of ligands and the conformation of the polymer. Three different complexes have been identified. The first (complex I), which is formed between pH 3 and 6, involves mainly the carboxyl groups of the polymer as ligands and is characterized by a strong absorption band at 238 nm. In this complex formation, the CD properties of hyaluronate do not charge appreciably. The second (complex II) forms between pH 6 and 8 bad shows a major change in CD properties. The changes include (1) a new positive CD band at 250 nm and a strong negative on in the π → π* amide transition region and (2) the disappearance of the negative n → π* amide CD band near 210 nm. A sharp increase in absorbance at 238 nm from complex I to II has been attributed to a conformational transition which is also manifested in the CD features of hyaluronate. Complex II involves, in addition to the carboxyl group, the nitrogen atom of the deprotonated acetamido group coordinated to Cu(II). The absorption at 230–280 nm is associated with the optically active charge-transfer transitions involving ligands to metal ion. At higher concentrations of the polymer or at higher pH, complex II aggregates to a gel, complex III. Chondroitin, differing from hyaluronic acid in the C-4 hydroxyl group configuration of the glucosamine moiety, does not show any CD change in the presence of Cu(II).The results provide further support to our fourfold helical structure of Cu(II)–hyaluronate complex at pH between 6 and 8. Intrinsic viscosities of hyaluronate in the presence of the cupric ion is lower than in the presence of other monovalent or bivalent cations, indicating a compact conformation of the polymer when it is complexed with Cu(II). 相似文献
5.
The effects of pH on hyaluronate as observed by light scattering 总被引:1,自引:0,他引:1
Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low-power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well-behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation. 相似文献
6.
Nuclear magnetic relaxation rates of H217O, 23Na+, and 25Mg2+ have been measured in aqueous hyaluronate solutions. The dependence on solution pH of the relaxation rates has been investigated, as well as the competition behavior of Na+ with Ca2+ and Mg2+. H217O and 23Na+ relaxation rates in chondroitin and hyaluronate solutions have been compared in the interval, 2 ? pH ? 12.5. The ion binding of hyaluronate can be fully accounted for by Coulomb interactions, with no need to involve chemical specificity. The hydration is only slighly pH dependent, and is comparable in magnitude to hydration of synthetic polyelectrolytes and monosaccharides. Ion-binding and hydration properties of hyaluronate and chondroitin are quite similar, except at elevated pH. At alkaline pH, an increase in charge density with pH is seen in hyaluronate and, to a much lesser degree, in chondroitin, possibly due to the titration of hydroxy groups. H217O data indicate an alkali-induced transition in both glycosaminoglycans. 相似文献
7.
Changes in conformation of hyaluronate and chondroitin sulfates following periodate oxidation were studied by CD. We monitored the progressive oxidation of these polymers by periodic acid at 4°C in pH 5.6 buffer. The negative CD band of hyaluronate at 208 nm decreased in intensity upon oxidation and changed its sign after 16 h of oxidation. In contrast, the 208-nm CD band of chondroitin sulfates decreased, but showed no change in sign even after 48 h of oxidation. A specific difference in solution conformation between hyaluronate and chondroitins may be responsible for the difference in oxidation-induced dichroic behavior. The results are discussed in terms of available x-ray diffraction analyses of these polymers. 相似文献
8.
T W Barrett 《Bio Systems》1976,8(3):103-109
The dynamic properties of hyaluronate solutions are discussed with relevance to some problems in sensory physiology (mechanoelectrical transduction), renal physiology, interstitial fluid regulation, and especially to the causes of open-angle glaucoma. With respect to the last problem: as recent biochemical evidence indicates that the hyaloid membrane does not exist, it now seems worthwhile to consider the increase in intraocular pressure present in the eye with glaucoma to be due--at least in the open-angle case--to a change in the specific gravity and hydrophilic nature of the hyaluronic acid in the vitreous body in particular, as well as in the trabecular meshwork. Densimetric experimental evidence indicates that the hyaluronate system could, indeed, produce the pressure changes seen in glaucoma, if intraocular pH changed but slightly. A hypothesis concerning the effect of acetazol amide on intraocular pressure is also presented. 相似文献
9.
Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures 总被引:4,自引:0,他引:4
Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds. 相似文献
10.
Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na+ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds. 相似文献
11.
Binding of bivalent cations by hyaluronate in aqueous solution 总被引:1,自引:0,他引:1
Furth G Knierim R Buss V Mayer C 《International journal of biological macromolecules》2008,42(1):33-40
The interaction between sodium hyaluronate and bivalent cations was investigated by conductometry, viscosimetry, circular dichroism and nuclear magnetic resonance spectroscopy. It is shown that the hyaluronate chains (Mn approximately 4.0 x 10(5)-1.7 x 10(6)g/mol) bind various bivalent cations (Ca2+, Mg2+, Mn2+, Fe2+, Cu2+, Zn2+, Cd2+ and Pb2+) at pH 6 in aqueous solutions. Hyaluronate deriving from Streptococcus equi was studied in comparison with dextran from Leuconostoc mesenteroides which was shown to develop no specific interactions with the bivalent cations. The molar relation between the bivalent cations and the disaccharide units of the resulting complex was determined with the result that one bivalent cation is bound by approximately five disaccharide units. Heavy metal ions (Cd2+, Pb2+) seem to bind stronger to the hyaluronate chain than their lighter counterparts (Ca2+, Mg2+). Circular dichroism spectra of the hyaluronate exhibit a cation-induced change in the n-pi* transition, indicating that the acetamide group of the aminoglucane unit is involved during the complexation. NMR spectra of hyaluronic acid in presence of paramagnetic manganese cations show strong interactions between the acetamide as well as the carboxylate groups and the cations. Based on these data, a structure of the binding complex is proposed which involves two disaccharide units. 相似文献
12.
Effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in cartilage explants.
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The effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in calf cartilage explants were examined. Pulse-chase experiments showed that conversion of low-affinity monomers to the high-affinity form (that is, to a form capable of forming aggregates with 1.6% hyaluronate on Sephacryl S-1000) occurred with a t1/2 of about 5.7 h in free-swelling discs at pH 7.45. Static compression during chase (in pH 7.45 medium) slowed the conversion, as did incubation in acidic medium (without compression). Both effects were dose-dependent. For example, the t1/2 for conversion was increased to about 11 h by either (1) compression from a thickness of 1.25 mm to 0.5 mm or (2) medium acidification from pH 7.45 to 6.99. Oscillatory compression of 2% amplitude at 0.001, 0.01, or 0.1 cycles/s during chase did not, however, affect the conversion. Changes in the hyaluronate-binding affinity of [35S]proteoglycans in these experiments were accompanied by no marked change in the high percentage (approximately 80%) of monomers which could form aggregates with excess hyaluronate and link protein. Since static tissue compression would result in an increased matrix proteoglycan concentration and thereby a lower intra-tissue pH [Gray, Pizzanelli, Grodzinsky & Lee (1988) J. Orthop. Res. 6, 777-792], it seems likely that matrix pH may influence proteoglycan aggregate assembly by an effect on the hyaluronate-binding affinity of proteoglycan monomer. Such a pH mechanism might have a physiological role, promoting proteoglycan deposition in regions of low proteoglycan concentration. 相似文献
13.
The influence of link-glycoproteins and mammalian lysozyme on the configuration and size of the hyaluronate molecule in highly diluted solutions under physiological electrolytic and pH conditions was investigated by light-scattering techniques and confirmed by column chromatography, isopycnic flotation, and boundary centrifugation. It was consistently found that link-glycoproteins induce an increase in the basic structural dimensions of the hyaluronate molecule in solution. It was also found that this increase was reversed or prevented under the action of mammalian lysozyme. Changes in configuration of the hyaluronate molecule could be related to its aggregating capacity when the hyaluronate interacts with proteoglycan subunits. It is postulated that link-glycoproteins induce structural changes in the hyaluronate molecule that might improve its aggregating capacity while mammalian lysozyme prevents or regulates such improvement. 相似文献
14.
Hyaluronate appears to be covalently linked to the cell surface 总被引:1,自引:0,他引:1
The purpose of this study was to examine the nature of the linkage between cell-surface hyaluronate and the plasma membrane. To accomplish this, rat fibrosarcoma cells were cultured in the presence of [3H]-acetate to isotopically label the hyaluronate, and then fixed with glutaraldehyde, which cross-links proteins but does not react directly with hyaluronate. The glutaraldehyde fixation stabilized the cells so that they could be manipulated in ways which would otherwise destroy cells. The fixed cells were then subjected to various treatments, and the amount of hyaluronate remaining on the cell surface was assayed via exhaustive digestion with Streptomyces hyaluronidase. Using this technique, we found that 1) cell-surface hyaluronate was quite stable for extended periods of time even in the presence of a large excess of non-labeled hyaluronate; 2) 4 M guanidine HCl and detergents did not extract a significant portion of cell-surface hyaluronate; 3) solutions of varying ionic strength (0-1 M NaCl) had no effect on the retention of hyaluronate; 4) the cell coat was stable in the range of pH 4-11, but outside this range a significant amount of hyaluronate was released; and 5) treatment with proteases released cell-surface hyaluronate. These results are consistent with the possibility that hyaluronate is covalently linked to a protein associated with the plasma membrane. Further support for this model came from experiments with the detergent Triton X-114, which can be used to separate soluble proteins from hydrophobic proteins. When nonfixed rat fibrosarcoma cells were extracted with this detergent and then partitioned by centrifugation, approximately 30 times as much hyaluronate was present in the detergent fraction which contained the hydrophobic proteins, as compared to the extracts pretreated with trypsin prior to phase separation. Again, these results suggest that cell-surface hyaluronate is directly linked to a hydrophobic core protein intercalated in the plasma membrane. 相似文献
15.
Three cutaneous propionibacteria, Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum, were grown in chemostats using semi-synthetic medium at various pH values. Growth occurred between pH 4.5 and 7.5 for P. acnes and pH 5.0 and 8.0 for P. avidum and P. granulosum. The highest mumax was at pH 6.0 for the three species. Maximum biomass production was obtained at pH 6.0 for P. acnes and P. avidum and at pH 7.5 for P. granulosum. Extracellular enzyme production occurred over the entire pH growth range when denaturation of the enzymes was taken into account. However, detectable activity of the enzymes was found in a narrower range of pH due to the denaturation of the enzymes at low or high pH values. The highest production of enzymes occurred at pH values between 5.0 and 6.0, apart from the production of hyaluronate lyase of P. granulosum (pH 6.0 to 7.0) and the proteinase of P. acnes and P. avidum (pH 5.0 to 7.5). Propionibacterium acnes produced a lipase, hyaluronate lyase, phosphatase and proteinase activity. Propionibacterium avidum produced a lipase and proteinase activity. Propionibacterium granulosum produced a lipase and hyaluronate lyase. 相似文献
16.
Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage 总被引:1,自引:0,他引:1
Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate. 相似文献
17.
Optical properties of hyaluronic acid. Ultraviolet circular dichroism and optical rotatory dispersion 总被引:3,自引:0,他引:3
The molar optical rotation at 220 nm and ellipticity values at 210 nm of both sodium hyaluronate and hyaluronic acid are greatly enhanced in comparison to the values for the monomeric units and oligosaccharides indicating a degree of preferred order. With increasing hydrogen ion concentration, there is no appreciable change in the 210 nm circular dichroic band, but the second circular dichroic band below pH 4 changes abruptly to the positive side and reaches a maximum value at pH 2·5. This positive circular dichroic band of hyaluronic acid is temperature and concentration dependent. The major change in sign and position of the second circular dichroic band of hyaluronic acid below pH 4 is attributed to the conformational change of a single polysaccharide chain or to a chain-chain interaction. The results indicate that increase in concentration or decrease in temperature and in the ionization of carboxyl group promotes the formation of ordered cross-link regions. The conformational changes found in solution have been interpreted as an order-disorder transition in the crosslink regions based on the interconversion of random coil and double helix. 相似文献
18.
Hyaluronate levels change dramatically during morphogenesis of various tissues and organs. Morphological detection of the exact temporal and spatial distribution patterns of hyaluronate may help to elucidate its role in morphogenesis. Since no specific direct method for visualizing hyaluronate with the light or electron microscope is currently available, we have developed a morphological probe by exploiting the high-affinity interaction of cartilage proteoglycan with hyaluronate. The core protein of this proteoglycan consists of a region that binds specifically to hyaluronate with a high association constant, and a region to which the majority of sulfated polysaccharide chains are covalently attached. The polysaccharide chains were removed by treatment with chondroitinase ABC, and the core protein, labeled with rhodamine, was used as the probe. This fluorescent probe binds reversibly and specifically to [3H]hyaluronate in a binding assay using ammonium sulfate precipitation of the core protein. The probe has been used to visualize the cell surface hyaluronate of rat fibrosarcoma cells, 3T3 cells, and SV-40 transformed 3T3 cells, three cell types with significantly different amounts of cell surface-associated hyaluronate. 相似文献
19.
T E Hardingham 《The Biochemical journal》1979,177(1):237-247
Proteoglycan fractions were prepared from pig laryngeal cartilage. The effect of link-protein on the properties of proteoglycan-hyaluronate aggregates was examined by viscometry and analytical ultracentrifugation. Aggregates containing link-protein were more stable than link-free aggregates at neutral pH, at temperatures up to 50 degrees C and in urea (up to 4.0M). Oligosaccharides of hyaluronate were able to displace proteoglycans from link-free aggregates, but not from the link-stabilized aggregates. Both types of aggregate were observed in the ultracentrifuge, but at the concentration investigated (less than 2 mg/ml) the link-free form was partially dissociated and the proportion aggregated varied with the pH and temperature and required more hyaluronate for saturation than did link-stabilized aggregate. The results showed that link-protein greatly strengthened the binding of proteoglycans to hyaluronate and suggest that under physiological conditions it 'locks' proteoglycans on to the hyaluronate chain. 相似文献