Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids

To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.     

Biosynthesis of hyaluronic acid by Streptococcus.

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.     

Purification and partial characterization of a novel hyaluronic acid-degrading enzyme from Antarctic krill (Euphausia superba)

Summary A novel enzyme degrading hyaluronic acid has been isolated, purified and characterized from Antarctic krill (Euphausia superba). A combination of affinity chromatography (Con A-Sepharose), gel filtration (Superose 6) and fast protein liquid chromatography (Mono Q) was used for the purification. The hyaluronidase activity was determined by a radial diffusion method based on hyaluronic acid incorporated into an agarose gel. Moreover, the beta-glucuronidase and endo-(1,3)-beta-D-glucanase activities were also followed through the process using phenolphtalein mono beta-glucuronic acid and laminarin as substrates. After the final purification step on Mono Q column, the chromatogram showed three main peaks designated A, B and C. Peak C contained high hyaluronidase activity undetectable in peak A and B. The betaglucuronidase activity was associated with peak A, while the endo-(1,3)-beta-D-glucanase activity was found in peak B and slight in peak C. The hyaluronidase was purified about 85-fold. It had a pH optimum of 5.3, a temperature optimum of 37°C and a molecular weight of 80 000 Daltons. On polyacrylamide gradient gel electrophoresis the enzyme fraction showed one major band associated with hyaluronic acid decomposition, slightly contaminated with a few other components. Isoelectric focusing in combination with a hyaluronic acid zymogram demonstrated one major band at pH 6.7 with high enzyme activity. Preliminary data on enzyme specificity suggest that krill hyaluronidase is a new endo-beta-glucuronidase and support the concept of krill enzymes as a remarkable and unusually effective digestive system adapted to the Antarctic marine ecosystem.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Cell-free synthesis of hyaluronic acid in Marfan syndrome.

The cell-free synthesis of hyaluronic acid has been demonstrated in extracts of cultured human fibroblasts. Preparations from fibroblasts of normal individuals as well as those from patients with Marfan syndrome incorporate glucuronic acid and N-acetylglucosamine from their UDP derivatives into hyaluronic acid. Extracts from Marfan fibroblasts demonstrate 3 to 10 times more total and specific hyaluronic acid synthetase activity than do preparations from normal fibroblasts. All synthetic activity was found in particulate fractions with the bulk of activity localized in material sedimenting as large membrane fragments. Marfan and normal preparations exhibited similar properties with respect to substrate, cofactor, pH requirements, and heat stability. Neither the Marfan nor normal enzyme systems could be stimulated by exogenous acceptors, nor did either preparation contain a soluble factor which stimulated or inhibited the enzymic activity of the other. The genetic defect in Marfan syndrome appears to result in increased activity of hyaluronic acid synthetase without demonstrable changes in properties of the particulate enzymes involved.     

Effect of trimethylcolchicinic acid on the synthesis and excretion of proteoglycans in tissue culture.

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.     

In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization.

Fetal tissue repair occurs without acute inflammation, prominent fibroplasia, or marked neovascularization. The fetal wound extracellular matrix is rich in hyaluronic acid (HA), while collagen is deposited in an organized normal dermal pattern. In various biologic systems, including regeneration and development, the controlled accumulation and subsequent degradation of hyaluronic acid is associated with distinct cellular and matrix events. Therefore, it is hypothesized that the abundance of hyaluronic acid in fetal wounds may influence cellular and/or matrix events such that tissue repair is highly organized and adult-like scarring does not occur. To test this hypothesis, the hyaluronic acid content of fetal rabbit wounds was reduced by specific degradation with Streptomyces hyaluronidase. Control wounds were treated with either enzyme buffer (n = 12) or denatured enzyme solution (n = 8) and exhibited a normal fetal healing response with scattered peripheral fibroblasts, a matrix of hyaluronic acid, and no infiltrating collagen. In marked contrast, the hyaluronidase-treated wounds (n = 14) demonstrated increased fibroblast infiltration, collagen deposition, and capillary formation. A significant reduction in the hyaluronic acid content of the hyaluronidase-treated wounds was confirmed biochemically. Since the degradation of hyaluronic acid resulted in an altered healing response, this study demonstrates that hyaluronic acid affects the cellular and matrix events in fetal healing and may be partially responsible for the unique qualities of this regenerative repair process.     

Kinetic studies on activation of peptide bond formation by hyaluronic acid.

1. In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction. 2. This reaction is activated by hyaluronic acid. 3. The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid. 4. The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator. 5. The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio.     

Degradation of Hyaluronic Acid, Poly- and Mono-Saccharides, and Model Compounds by Hypochlorite: Evidence for Radical Intermediates and Fragmentation

Degradation of hyaluronic acid by oxidants such as HO^· and HOCl/ClO⁻ is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO^· has been investigated extensively, reaction with HOCl/ClO⁻ is less well defined. Thus, little is known about the site(s) of HOCl/ClO⁻ attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO⁻ with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220–340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO⁻ reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R—NCl—C(O)—R′) species, which decompose rapidly to give radicals via either homolysis (to produce N· and Cl·) or heterolysis (one-electron reduction, to give N· and Cl⁻) of the N—Cl bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO⁻ with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO⁻ concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.     

Glycosaminoglycans of solid and ascites forms of the P1798 murine lymphosarcoma

Total glycosaminoglycan levels are similar from subcutaneous, ascites and mesenteric forms of the P1798 lymphosarcoma. However, P1798 cells implanted subcutaneously produce a solid tumor rich in hyaluronic acid with lesser amounts of chondroitin 4-/6-sulfates as well as an undersulfated species of chondroitin sulfate, while cells implanted intraperitoneally produce ascites tumors which generate hyaluronic acid, almost exclusively. Mice bearing advanced ascites tumors develop solid mesenteric tumors which exhibit chondroitin 4-/6-sulfates and under-sulfated chondroitin sulfate in addition to hyaluronic acid.     

Evaluating specific adhesion of Plasmodium falciparum-infected erythrocytes to immobilised hyaluronic acid with comparison to binding of mammalian cells

A feature of infection with Plasmodium falciparum is the ability of parasite-infected erythrocytes to adhere to vascular endothelial cells and accumulate in vital organs, associated with severe clinical disease. Hyaluronic acid was recently identified as a receptor for adhesion and has been implicated in mediating the accumulation of parasites in the placenta. Here, we report in vitro assays to measure specific adhesion of infected erythrocytes to hyaluronic acid that is distinct from binding to chondroitin sulphate A, another glycosaminoglycan implicated as a receptor in placental malaria. In this study, specific adhesion of mature stage infected erythrocytes to hyaluronic acid of high purity immobilised on plastic surfaces was abolished by pre-treating hyaluronic acid with a specific hyaluronate lyase from Streptomyces, whereas the same treatment of chondroitin sulphate A had little effect. Adhesion to hyaluronic acid could not be explained by the presence of chondroitin sulphate A or other glycosaminoglycans in the hyaluronic acid preparations. Chinese hamster ovary cells bound in a similar manner in the assays and confirmed that hyaluronic acid was appropriately immobilised for cell adhesion. In contrast to parasites, these cells did not adhere to chondroitin sulphate A. The adsorption of hyaluronic acid onto plastic surfaces was also confirmed by the use of a specific hyaluronic acid-binding protein. Fixing cells with glutaraldehyde at the completion of adhesion assays reduced the number of parasites remaining adherent to hyaluronic acid, but not to chondroitin sulphate A or CD36. These findings have important implications for understanding and evaluating interactions between P. falciparum and hyaluronic acid that may be involved in disease pathogenesis.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, a glycoprotein interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. The primary structure of the link protein has been determined with a view to defining its interaction with both hyaluronic acid and proteoglycan. Thus, the link protein has been digested with staphylococcal V8 protease, trypsin, and chymotrypsin and the resulting peptides characterized by amino acid composition and sequence. We have determined that the link protein is a single peptide with 339 amino acid residues. The protein core has a molecular weight of 38,564. There is one N-linked oligosaccharide at residue 41 with a molecular weight of approximately 2,500. There are five disulfide bonds which define three loops within the amino acid sequence. The loop nearest to the NH2-terminal contains 78 amino acids and is followed by a section of 42 amino acids between it and the second loop. The second and third loops display considerable homology with each other; they consist of 71 and 70 amino acids, respectively, each contain two disulfide bonds, and both loops possess, approximately centrally, an epitope for the species nonspecific anti-link protein monoclonal antibody, 8A4. These loops are separated by a short section of 27 amino acids. We speculate that these loops are functionally important in the interaction of link protein with hyaluronic acid, as they appear to be the most conserved regions of link protein between species.     

Isolation of lipid glucuronic acid and N-acetylglucosamine derivatives from a rat fibrosarcoma

Incubation of labeled UDP-GlcUA and UDP-GlcNAc with microsomes of a fibrosarcoma yielded labeled glycolipids resistant to hydrolysis with dilute alkali. These compounds have been tentatively identified as lipid-GlcNAc, lipid-GlcNAc-GlcUA and lipid-tetra and hexasaccharides containing both GlcUA and GlcNAc.     

Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.     

The hydrodynamic resistance of hyaluronic acid: estimates from sedimentation studies

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.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system

In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...