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1.
Fractionation of a hyaluronic acid preparation in a density gradient. Some properties of the hyaluronic acid 总被引:2,自引:2,他引:0
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1. Hyaluronic acid was isolated from ox synovial fluid by sedimentation equilibrium in a caesium chloride density gradient (Silpananta, Dunstone & Ogston, 1967). The product was almost free from chondroitin sulphate and from protein. 2. Its composition did not differ significantly from that of the carbohydrate part of the protein-containing material isolated by filtration. Its physicochemical properties and molecular configuration were similar, except for its viscosity, which showed markedly reduced concentration-dependence and shear-dependence. This suggests that the associated protein tends to form links between molecules of hyaluronic acid. 3. The accurate measurement of viscosity at very low velocity gradient, by use of the damping of oscillations in a Couette viscometer, is described. 4. A method is described for measuring, approximately, the thermodynamic non-ideality of a solute from the shape of its schlieren curve at sedimentation equilibrium in a density gradient. 5. A value for the partial specific volume of hyaluronic acid in dilute salt solution was calculated from its isopycnic density in a caesium chloride gradient. 相似文献
2.
Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products. 相似文献
3.
Fractionation of a hyaluronic acid preparation in a density gradient: The isolation and identification of a chondroitin sulphate 总被引:7,自引:5,他引:2
1. Following the suggestion of Nichol, Ogston & Preston (1967) that hyaluronic acid, prepared by filtration from ox synovial fluid, contains a component of high density, such material has been detected and isolated by equilibrium sedimentation in a density gradient of caesium chloride. 2. This material (fraction III) has been characterized as a chondroitin sulphate-protein complex of average molecular weight about 250000. Its amino acid pattern is characteristic of such complexes present in cartilage. It contains a proportion of glucosamine (as well as galactosamine); this is not due to contamination with hyaluronic acid. 3. Preliminary findings on fraction I (low density) and fraction II (intermediate density) suggest that these consist chiefly of protein and hyaluronic acid respectively. 相似文献
4.
Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains. 相似文献
5.
C R Ethier 《Biorheology》1986,23(2):99-113
The problem of estimating the permeability of hyaluronic acid as a function of concentration has been examined. A previously known method of obtaining permeability from sedimentation studies has been employed, and the results have been compared to those obtained from convection studies, in which solvent is forced through the hyaluronic acid. The two sets of results were seen to be at variance, which could be explained by a flow-induced polarization of the hyaluronic acid in the convection studies. The polarization phenomenon was described in terms of a convection-associated compaction balancing the hyaluronic acid's intrinsic resistance to compression. Based on these results and other arguments, it was suggested that data from sedimentation studies provide a more accurate estimate of hyaluronic acid permeability than do convection experiments. The implications of this finding in a physiological context were briefly discussed. 相似文献
6.
Crescenzi V Francescangeli A Taglienti A Capitani D Mannina L 《Biomacromolecules》2003,4(4):1045-1054
Hydrophilic networks based on functionalized hyaluronic acid and on partially acetylated chitosan, respectively, have been obtained. In the case of hyaluronic acid (HA), primary amino functionalities have been introduced along the polysaccharide chains. The ensuing derivatives, i.e., HA-lysine (HA-K), HA-diamino pentane (HA-DAP), and HA-glycine-lysine (HA-GK), have been characterized by high field NMR spectroscopy. NMR 2D-DOSY experiments have allowed us to optimize the purification procedure. Chitosan was made soluble in water by partial acetylation. Cross-linking reactions have been performed using glutaraldehyde. The obtained networks have been qualitatively characterized by means of (13)C CP-MAS NMR technique. The hydrogels have been characterized also in terms of water uptake. 相似文献
7.
Analysis of protein-protein interactions in highly concentrated solutions requires a consideration of the non-ideality in such solutions which is expressed by the virial coefficients. Different equations are presented to estimate effects of the thermodynamic non-ideality on the macromolecular interaction of self-associating proteins in sedimentation equilibrium experiments. Usually the influence of thermodynamic non-ideal behavior are described by concentration power series. The convergence of such power series is limited at high solute concentration. When expressing the thermodynamic non-ideality by an activity power series this disadvantage can be minimized. The developed centrifuge equations are the basis for a global analysis to estimate equilibrium constants and the corresponding thermodynamic activities of the reactants. Based on fit analysis of synthetic concentration profiles it was established that marked deviations from the expected association constants are observed for proteins with strong association forces between solute molecules. Considerable differences were also observed in weakly interacting systems. This was due to the excluded volume of the protein which is similar in magnitude to the binding constant. For interactions with moderate affinities values extremely close to the true binding values were obtained, as confirmed by experimental results with concanavalin A. 相似文献
8.
The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma 总被引:15,自引:11,他引:4
1. Materials containing hyaluronic acid have been prepared by filtration (Ogston & Stanier, 1950) from ox synovial fluid and from a protein-rich human mesothelioma fluid. The ox material has been deproteinized by treatment with chloroform and pentanol and by gradient elution on DEAE-Sephadex; several fractions were obtained by the latter method. These materials can be stored in solution at −20° without change of properties. The ox material contained 21% of protein; all other preparations contained less than 6% of protein. 2. The two materials have been compared by sedimentation and viscosity and shown to be closely similar. Treatment of the ox material with neuraminidase caused no change in its viscosity behaviour. 3. Information about the molecular configuration of the ox material has been obtained from measurements of light-scattering and viscosity. The results, though consistent with a highly extended configuration, are not consistent with a linear random-coil configuration. It is tentatively suggested that the structure may have some degree of branching and of cross-linking, which give it a rigidity with respect to expansion of the molecular domain that would not be possessed by a random coil. 4. The deproteinized material recovered from DEAE-Sephadex, though polydisperse, showed unchanged average molecular weight; however, the average radius of gyration was greater than before this treatment. 5. Acidification to approx. pH3 resulted in a contraction of the structure, with only a slight degree of expansion when the pH was restored to 6·8–7·0. 6. Measurements of optical rotatory dispersion qualitatively support a structure less simple than a linear random coil. 7. Colloid osmotic pressures of mixed solutions of bovine serum albumin and of hyaluronic acid prepared by filtration from ox synovial fluid have been measured. The results agree approximately with those of Laurent & Ogston (1963) but are in quantitative disagreement with the partition measurements of Ogston & Phelps (1960). The relationships between thermodynamic quantities in a quaternary system of electrolytes are discussed in Appendix 2. 8. Refractometric measurements have been made in connexion with light-scattering measurements, as the basis for a convenient method of determining the concentrations of solutions of hyaluronic acids, and to measure the partition of sodium chloride in dialysis experiments. The theory of the last use is discussed in Appendix 1. 9. Sedimentation measurements on the ox preparation have been made up to a concentration of 1·4×10−2g./ml. The form of the sedimentation coefficient–concentration relationship is discussed. The value of the sedimentation coefficient at higher concentration is the basis of an illustration of the likely effect of hyaluronic acid on the flow of water through narrow channels in connective tissue. 10. Available colorimetric methods have been shown to give low estimates for glucuronic acid when applied to highly polymerized materials, as compared with estimates by decarboxylation. A spectrophotometric titration with cetylpyridinium bromide has been shown to give estimates of carboxyl groups that agree well with those of decarboxylation when applied to preparations of hyaluronic acid under suitable conditions; the results are not affected by the presence of protein. 11. Estimates of glucosamine (Ogston, 1964) have been found to be low compared with those of total acetyl, independently of the presence of protein. The magnitude of the discrepancy is characteristically different for preparations from ox synovial fluid and from mesothelioma. 12. Sialic acid was estimated in several preparations. It is likely that this forms part of the protein. 13. Analyses of preparations for total nitrogen, amino acids, total acetyl, glucuronic acid (by decarboxylation) and ash account for at least 95·7% of the dry weight in terms of N-acetylglucosaminyl, glucuronyl, protein and metal ions. Previously published analyses of hyaluronic acids are reviewed. 14. The estimated molar ratios of glucuronic acid to glucosamine were all significantly greater than unity. 15. The analytical results are interpreted as agreeing with the physicochemical measurements in suggesting a more complex structure, for at least some hyaluronic acids, than that of an alternate linear copolymer in random-coil configuration. 相似文献
9.
The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected. 相似文献
10.
The action of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans has been studied on the L cell strain. The incorporation of precursors has been measured, and proteoglycans produced in the culture medium have been extracted and their concentration determined. The mucopolysaccharide components have been studied by electrophoresis. Control cultures produce hyaluronic acid, dermatan sulfate and very low concentrations of chondroitin 4-sulphate or 6-sulphate. Cultures treated with trimethycolchicinic acid (4 mu g/ml) produce hyaluronic acid, very high concentrations of chondroitin 4-sulphate or 6-sulphate and only traces of dermatan sulphate. So, trimethylcolchicinic acid does not modify the synthesis of hyaluronic acid: it considerably increases the production of chondroitin 4-sulphate or 6-sulphate and inhibits the production of dermatan sulphate. Protein fraction of the proteoglycans is proportionally increased in treated cultures, but there is no marked difference between amino acid concentrations of proteoglycans extracted from control and treated cultures. A slight fall in the cystine concentrations was the only change in the amino acid content of proteoglycans extracted from treated cultures. A hypothesis to explain these results is discussed. 相似文献
11.
David A. Swann 《The Biochemical journal》1969,114(4):819-825
Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1.2x10(6) and 6.5x10(4) respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid. 相似文献
12.
Circular dichroism (CD) studies have been made on hyaluronic acid (HA) obtained from dialyzed owl monkey vitreous. The protein content of these samples is low enough not to interfere with the CD measurements of hyaluronic acid. The ellipticity values of vitreous HA are higher than those of HA from other tissues, indicating a higher degree of preferred order. Since the purification procedures involve only dialysis, the owl monkey vitreous can be a model tissue for structural studies of HA close to its native state. 相似文献
13.
From expressions derived for the space-filling effects of small inert solutes on kinetic parameters for univalent enzymes undergoing isomerizations that are substrate-induced and pre-existing, it is concluded that experimental observation of an enhanced maximal velocity in the presence of inert solute can only reflect the existence of the former type of conformational change; and that the isomerization must be governed by a relatively small equilibrium constant. Similar conclusions apply to multivalent enzymes exhibiting Michaelis-Menten kinetics. Extension of the theory to provide quantitative expressions for multivalent enzymes has made possible the numerical simulation of thermodynamic non-ideality effects on systems conforming with the Monod and Koshland models of allostery. In that regard the simulated Scatchard plots for the two models differ sufficiently in form to suggest that detailed examination of the space-filling effects of small solutes on the kinetics of an allosteric enzyme may, under favourable circumstances, allow identification of the appropriate allosteric mechanism. Finally, these considerations of thermodynamic non-ideality in relation to the kinetics of allosteric enzymes have revealed formal similarities between the consequences of space-filling by inert solutes and the specific effects of allosteric activators or inhibitors. Attention is drawn to the possible implications of this observation in relation to the functioning of allosteric enzymes in vivo, where catalytic performance may be modified by factors no more specific than the ability of unrelated solutes to occupy space in the highly concentrated cellular environment. 相似文献
14.
Sannino A Madaghiele M Conversano F Mele G Maffezzoli A Netti PA Ambrosio L Nicolais L 《Biomacromolecules》2004,5(1):92-96
The aim of this work is to obtain a chemically cross-linked hydrogel from hyaluronic acid and cellulose derivatives that exhibits sensitivity to variation of the composition of the external absorbing medium and an equilibrium sorption capacity higher than a common hyaluronic acid-based hydrogel, in view of its potential use in prevention of postsurgical soft tissue adhesion. This has been achieved by chemical stabilization of hyaluronic acid (HA) and cellulose derivatives, hydroxyethylcellulose (HEC) and carboxymethylcellulose (CMCNa) through the difunctional cross-linker divinyl sulfone. Significant increase in sorption capacity, both in water and in water solutions at different ionic strength, has been observed for these samples in comparison with hydrogels obtained through chemical stabilization of hyaluronic acid. Moreover, different dehydration procedures adopted for the xerogel synthesis have been used, which resulted in a modulation of the equilibrium sorption capacity. Hyaluronic acid stability has been confirmed by means of NMR analysis. 相似文献
15.
Rabbit antibodies to degraded and intact glycosaminoglycans which are naturally occurring and present in arthritic rabbits 总被引:3,自引:0,他引:3
A R Poole A Reiner P J Roughley B Champion 《The Journal of biological chemistry》1985,260(10):6020-6025
Using enzyme-linked immunosorbent assays and radioimmunoassays employing chondroitinase ABC-treated rabbit cartilage proteoglycan, we have shown that approximately one-third of the outbred New Zealand white rabbits we have examined possess naturally occurring antibodies which react with oligosaccharides of hyaluronic acid (independently of chain length) bearing saturated and 4,5-unsaturated glucuronosyl residues at the nonreducing ends. Such antibodies were also found in a similar proportion of rabbits with an experimental inflammatory arthritis. There was a preferential reactions in the majority of sera with unsaturated oligosaccharides of hyaluronic acid. One serum (R64) reacted only with unsaturated oligosaccharides of hyaluronic acid. Sera reacted also with unsaturated (never saturated) oligosaccharides of chondroitin 4-sulfate and with chondroitin 6-sulfate, particularly when chondroitin sulfate oligosaccharides remained bound to a proteoglycan core protein. Reactions were also observed to both unsaturated and saturated oligosaccharides of chondroitin. Some of these sera also reacted with intact hyaluronic acid and chondroitin but never with intact chondroitin sulfate. The antibodies were present in the IgG fraction of four sera studied and in the IgM fraction of one of these sera: they bound through the F(ab')2 region of the molecule. These observations suggest that, in some rabbits, humoral immunity to hyaluronic acid and/or chondroitin sulfate bound to core protein can develop after these reactive glycosaminoglycans have been degraded by eliminases or hydrolases produced by naturally occurring bacteria and rabbit cells, respectively. Immunological studies of proteoglycans and hyaluronic acid treated with eliminases and hydrolases employing rabbit antisera, and possibly those from other species, should be evaluated in the light of these observations. 相似文献
16.
The disorder of hyaluronic acid metabolism in cultured skin fibroblasts derived from a patient with the Hurler syndrome 总被引:3,自引:2,他引:1
The metabolism of hyaluronic acid in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was examined. 1. An increased net incorporation of [(3)H]glucose into the hyaluronic acid fraction of the Hurler-syndrome cells occurred when compared with normal cells. 2. During a ;chase' period, approx. 35% of the radioactivity derived from glucose was lost from the hyaluronic acid fraction of the Hurler-syndrome cells, whereas the normal cells retained all their radioactivity. 3. Although the Hurler-syndrome cells contained a ninefold greater amount of hyaluronic acid than normal cells, simultaneous determination of the specific radioactivity derived from the label revealed a value for the Hurler-syndrome cells one-half that of normal cells. These results are taken to indicate that the Hurler cells synthesize hyaluronic acid de novo at a higher rate than do normal cells. 4. Exposure of Hurler-syndrome cultured fibroblasts to a crude urine corrective-factor preparation (Neufeld & Cantz, 1971), now known to contain alpha-l-iduronidase, the specific Hurler-syndrome corrective factor (Bach et al., 1972), decreased the hyaluronic acid content to near-normal values before any effect was observed on [(3)H]glucose incorporation into the hyaluronic acid fraction. 5. In addition, the hyaluronic acid content of the normal cells decreased after exposure to the corrective factor of urine. 6. The mobilization of hyaluronic acid in Hurler-syndrome and normal cells exposed to the crude corrective-factor preparation of urine caused a decrease in specific radioactivity in the ;corrected' Hurler-syndrome cells and an increase in specific radioactivity in the ;corrected' normal cells. 相似文献
17.
F.Ross Hallett A.Laurence Gray 《Biochimica et Biophysica Acta (BBA)/General Subjects》1974,343(3):648-655
Autocorrelation functions for the intensity of laser light scattered from solutions of hyaluronic acid have been measured under a variety of experimental conditions. The form of these functions is consistent with the large amount of long-range intra- and intermolecular interactions characteristic of polyelectrolyte solutions. Further experiments have been performed in order to study the effect of hyaluronic acid on the Brownian diffusion of polystyrene spheres. Using several different sphere sizes as solution probes, the importance of the ionic environment in determining hyaluronic confirmation has been demonstrated. 相似文献
18.
Hyaluronic acid synthesis and secretion by rat liver fat storing cells (perisinusoidal lipocytes) in culture 总被引:2,自引:0,他引:2
The ability of rat liver fat storing cells to synthesize and to secrete hyaluronic acid was examined in monolayer cultures. The cells produce [3H] glucosamine-labeled hyaluronic acid, of which about 80% are secreted into the medium. The synthesis rate per cell (mg DNA) of labeled total glycosaminoglycans and hyaluronic acid in the medium increases significantly with culture time, but hyaluronic acid expressed as fraction of total glycosaminoglycans declines from about 0.70 in early cultures (up to the 4th day) down to 0.20 in advanced cultures. Cycloheximide increases and beta-D-xylopyranoside decreases significantly the fraction of hyaluronic acid in the medium, colchicine up to 5 microM was without effect. The synthesis of hyaluronic acid is a newly recognized function of this special type of sinusoidal liver cells. The results suggest that fat storing cells are likely to be a major source of hyaluronic acid in normal and probably also in injured liver. 相似文献
19.
This investigation re-examines theoretical aspects of the allowance for effects of thermodynamic non-ideality on the characterization of protein self-association by frontal exclusion chromatography, and thereby provides methods of analysis with greater thermodynamic rigor than those used previously. Their application is illustrated by reappraisal of published exclusion chromatography data for hemoglobin on the controlled-pore-glass matrix CPG-120. The equilibrium constant of 100/M that is obtained for dimerization of the alpha(2)beta(2) species by this means is also deduced from re-examination of published studies of concentrated hemoglobin solutions by osmotic pressure and sedimentation equilibrium methods. 相似文献
20.
This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm?3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied.The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of ? 197 cal deg. mol?1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation.Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔGO ? ?3 k cal mol?1, ΔHO ~ 0, ΔSO ~ 10 cal deg.?1 mol?1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin. 相似文献