Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
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PMID:Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. 76

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase, nucleases (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowes 1-X2 (Cl-) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2-0.3 microgram hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (OR C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.
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PMID:Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver. 90 87

L-[14C]Iduronic acid-containing sulfated galactosaminoglycans were formed by incubation of a fibroblast particulate fraction with UDP-D[14C]glucuronic acid, UDP-N-acetylgalactosamine, and sulfate donor (3'-phosphoadenylylsulfate). The formation of L-iduronic acid was strongly promoted by concomitant sulfation of the polymer. In the absence of sulfate donor 5 to 10% of the [14C]uronic acid residues were L-iduronic acid. However, when 3'-phosphoadenylylsulfate was included in the incubation mixture the amount of L-iduronic acid in the product increased 3 to 5-fold. Furthermore, approximately the same quantity of L-[14C]iduronic acid was recovered from the product formed in a pulse-chase experiment where incorporation of 14C-isotope preceded sulfation. It was therefore concluded that C-5 inversion of D-glucuronic acid to L-iduronic acid occurred on the polymer level as shown previously for the biosynthesis of heparin (Hook, M., Lindahl, U., Backstrom, G., Malmstrom, A., AND Fransson, L-A., J. Biol. Chem. (1974) 249, 3908). This conclusion was supported by the finding that no L[14C]iduronic acid could be detected in the UDP-hexuronic acid pool during this experiment. Nonsulfated and sulfated [14C]galactosaminoglycan products were degraded separately with chondroitinase-AC. The non-sulfated products afforded primarily disaccharide and a small amount of tetrasaccharide, while the sulfated products yielded, in addition, a considerable amount of larger oligosaccharides. Tetrasaccharides from nonsulfated products contained L-iduronic acid indicating that C-5 inversion at solitary sites can occur in the absence of sulfation of adjacent hexosamine moieties. The larger oligosaccharides obtained after chondroitinase-AC digestion of sulfated products yielded L-iduronic acid upon acid hydrolysis and were susceptible to chondroitinase-ABC digestion. The split products were almost exclusively 4-sulfated disaccharides. These results demonstrate that formation of blocks of L-iduronic acid-containing repeat periods is associated with 4-sulfation of adjacent hexosamine moieties.
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PMID:Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues. 112 48

Proteoglycans of bovine compact bone were purified by chromatography of the formic acid precipitate of an EDTA extract. The sequential chromatographic steps consisted of gel filtration on Sepharose CL-6B in 4-M guanidine HCl, ion-exchange chromatography on DEAE-Sephacel in 4-M urea and rechromatography on Sepharose CL-6B in 4-M guanidine HCl. The preparation consisted of a relatively small proteoglycan (Kav = 0.4 on Sepharose CL-6B) containing about 40% protein, 21% hexuronic acid, 23% galactosamine and lesser amounts of other monosaccharides. The core protein was shown by gradient NaDodSO4 gel electrophoresis, electrotransfer and immunodetection to be monodispersed with an Mr = 45,000. Analysis of glycopeptides obtained after papain digestion of the proteoglycan and separation from glycosaminoglycan chains by gel chromatography, indicated that both N-linked and O-linked oligosaccharides were present. The glycosaminoglycan chains liberated by papain digestion eluted from Sepharose CL-6B as a broad peak with Kav = 0.50, slightly ahead of the position of elution of bovine nasal cartilage glycosaminoglycans (Kav = 0.52); the bone glycosaminoglycans are thus slightly larger than those from cartilage and smaller than the ones attached to fetal bone proteoglycans. These chains were totally susceptible to chondroitinase AC II, a procedure that yielded unsaturated disaccharides corresponding predominantly to chondroitin-4-sulfate, and to a lesser extent chondroitin-6-sulfate. Antisera raised against adult bone proteoglycans cross-reacted with core protein of bone proteoglycan (obtained after chondroitinase digestion) but not with papain digested proteoglycan. In addition, they cross-reacted with core protein and trypsin-liberated, chondroitin sulfate rich region (AlTAl) derived from cartilage proteoglycans and, to a lesser extent, rat bone proteoglycans. No cross-reactivity could be detected to Smith-degraded cartilage proteoglycans, bone acidic glycoproteins or serum proteins.
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PMID:Proteoglycans of adult bovine compact bone. 293 15

The oversulphated galactosaminoglycans synthesized by rat mucosal mast cells were isolated from the small intestine of animals infected with the nematode Nippostrongylus brasiliensis, which causes proliferation of these cells. The 35S-labelled polysaccharides were degraded by digestion with chondroitinase ABC, and the structures of the disaccharide products were determined by cleavage with mercuric acetate followed by electrophoretic characterization of the resultant sulphated monosaccharides. It was concluded that about half of the disulphated disaccharide units in the polysaccharide consisted of chondroitin sulphate E-type structures [GlcA-GalNAc(4,6-di-OSO3)], in which both sulphate groups were located on the N-acetylgalactosamine unit. The remainder consisted of isomeric structures with one sulphate group on the N-acetylgalactosamine residue and one on the hexuronic acid unit and presumably represented the dermatan sulphate-type sequence [IdoA(2-OSO3)-GalNAc(4-OSO3)].
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PMID:Identification of oversulphated galactosaminoglycans in intestinal-mucosal mast cells of rats infected with the nematode worm Nippostrongylus brasiliensis. 317 41

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle. 360 17

The major urinary trypsin inhibitor (Mr 44 000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26 000) but still inhibits trypsin.
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PMID:The major human urinary trypsin inhibitor is a proteoglycan. 373 76

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.
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PMID:The proteoglycans of the canine intervertebral disc. 392 Oct 57

Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.
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PMID:Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin. 772 74

Variable substitutions and locations of the sulfate esters along the backbone of chondroitin/dermatan sulfate chains, combined with their carbohydrate structures, present topographies to immune systems which can be recognized as antigenic. This has led to the development of a number of monoclonal antibodies which recognize distinct epitopes in the native structures of these glycosaminoglycan chains. In some studies, the original chondroitin/dermatan sulfate proteoglycan was digested with chondroitinase enzymes before being used as an immunogen. in this case, the linkage oligosaccharides remaining bound to the core protein contain a modified (4,5-unsaturated) hexuronic acid derivative at their non-reducing ends as a result of the eliminase mechanism of the enzyme. This 'haptenic' structure is highly antigenic and has led to the development of a number of monoclonal antibodies which recognize this structure as part of their epitopes. Examples of the use of some of these monoclonal antibodies for localization of proteoglycan structures in tissue sections and on transblots are described. The precise structures are known for only a few of the native epitopes recognized by these monoclonal antibodies. Recent analytical methods have been developed for determining structures of chondroitin sulfate oligosaccharides. An example of the use of these methods to analyze the structures of the non-reducing termini of chondroitin/dermatan sulfate chains is discussed. The results show their potential value for quantifying the native epitope recognized by a monoclonal antibody, designated 3B3, which recognizes chains terminated by glucuronic acid-N-acetylgalactosamine-6-sulfate. Such methods should be useful for determining the epitope structures for other monoclonal antibodies in this class.
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PMID:Immunology of chondroitin/dermatan sulfate. 859 49


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