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)

Fibroblasts in culture were incubated with [35S]sulfate/[3H]glucosamine or [35S]sulfate/[3H]leucine. Proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer or from a trypsin digest of the cells and an extract of the cell residue. Proteoglycans were isolated by density gradient centrifugation, gel permeation, and ion exchange chromatography after digesting contaminating proteogalactosaminoglycans with chondroitinase ABC. Gel chromatography suggests that the cell-derived protoheparan sulfate had an Mr = 350,000 whereas the trypsin-released and the medium-derived counterparts both had an Mr = 140,000. Reduction and alkylation of the cell-derived proteoglycan gave rise to a component with Mr = 140,000, whilst the medium-derived form was not affected. Degradation of cell-associated proteoheparan sulfate by trypsin followed by papain or alkali suggest that the core protein consists of three types of regions, heparan sulfate-containing regions of Mr = 140,000, oligosaccharide-containing regions, and nonglycosylated peptide regions containing most of the [3H]leucine. The heparan sulfates of the cell- and medium-derived proteoglycans were similar in size distribution and charge density and with regard to the proportions and arrangements of various building blocks.
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PMID:Proteoheparan sulfate from human skin fibroblasts. Isolation and structural characterization. 622 77

Involvement of covalently linked protein or peptide in the structure or synthesis of hyaluronate has not previously been convincingly demonstrated. We have developed conditions for double-labeling with [3H]leucine and [14C]acetate, then isolating and characterizing the cell-associated and secreted hyaluronate-protein complexes of Rous sarcoma virus-transformed chick embryo fibroblasts. The preparations were purified by Bio-Gel A-15m gel filtration and CsCl density gradient ultracentrifugation under dissociative conditions, followed by acid agarose gel electrophoresis in the presence of 0.1% Nonidet P-40. The purified hyaluronate preparations did not change their 3H:14C ratios after further sodium dodecyl sulfate or alkali treatment. The cell-derived hyaluronate-protein was resistant to pronase but susceptible to proteinase K in the presence of sodium dodecyl sulfate. After chondroitinase ABC digestion, the cell-derived 3H-labeled protein was separated from the 14C-labeled hyaluronate disaccharides, then shown to give a broad band corresponding to Mr approximately 12,000 on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and to be susceptible to both pronase and proteinase K. The corresponding 3H-labeled peptide was prepared in the same manner from the medium hyaluronate and the [3H]leucine shown to be present in material smaller in amount and size than that from the cell. We propose from these and other published data that the cell-associated hyaluronate-protein may be bound to the cell surface and that the hyaluronate in the medium may be derived from it as a result of proteolytic scission.
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PMID:Hyaluronate-protein complex of rous sarcoma virus-transformed chick embryo fibroblasts. 626 45

Retinas were labeled in culture with [3H]glucosamine or [3H]leucine and [35S]sulfate and extracted sequentially with physiologically balanced saline and 4 M guanidine HCl. They were dialyzed into associative conditions (0.5 M NaCl) and chromatographed on agarose columns. Under these conditions, some of the proteoglycans were associated in massive complexes that showed low buoyant densities when centrifuged in CsCl density gradients under dissociative conditions (4 M guanidine HCl). Much of the label in these complexes was in molecules other than proteoglycans. Most of the proteoglycans, however, were included on the agarose columns, where they appeared to be constitutionally of low buoyant density. They resisted attempts to separate potential low buoyant density contaminants from the major proteoglycans by direct CsCl density gradient centrifugation or by the fractionation of saline or 8 M urea extracts on diethylaminoethyl-Sephacel. The diethylaminoethyl-Sephacel fractions were either subjected to CsCl density gradient centrifugation or were chromatographed on Sephacryl S-300, in both cases before and after alkaline cleavage, to confirm the presence of typical O-linked glycosaminoglycans. The medium and balanced salt extracts were enriched in chondroitin sulfate and other sulfated macromolecules, possibly highly sulfated oligosaccharides, that resisted digestion by chondroitinase ABC but were electrophoretically less mobile than heparan sulfate. Guanidine HCl or urea extracts of the residues were mixtures of high and low density proteoglycans that were enriched in heparan sulfate.
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PMID:Low buoyant density proteoglycans from saline and dissociative extracts of embryonic chicken retinas. 636 28

Many melanoma-associated antigens have been identified by monoclonal antibodies. One of these monoclonal antibodies, O1-94-45, binds only to melanomas, nevus cells, some astrocytomas, and fetal epitheloid cells. There are approximately 100,000 cell surface antigens per melanoma cell with an association constant of 3 X 10(8) M-1. The antigen is efficiently extracted from the membrane only in the presence of detergent and is, therefore, bound by hydrophobic forces. However, it is also shed into the culture supernatant during normal cell growth. The two components of the O1-95-45 antigen are a chondroitin sulfate proteoglycan (CSP, greater than 500,000 Da) and a glycoprotein gp260 (260,000 Da, pI 6.9). CSP contains chondroitin sulfate and N-linked and O-linked oligosaccharides. Only N-linked saccharides were associated with gp260. The antigenic site is expressed on both components and is heat-sensitive. Since the CSP was converted to gp260 by chondroitinase, the protein cores of the two molecules are the same or similar. For more detailed study the O1-95-45 antigen was purified by immunoaffinity chromatography. The amino acid composition of the purified antigen was relatively polar with an unusually high Leu content and low Lys content. Initial attempts to sequence the antigen were unsuccessful probably due to a blocked N-terminus. CSP and gp260 were partially separated by gel filtration chromatography, and both were found to carry the O1-95-45 antigenic determinant. Three other monoclonal antibodies were found to bind the purified antigen at a site or sites different from the O1-95-45 epitope and one other monoclonal antibody may bind at the same site. Two of these antibodies were used for a double determinant immunoassay.
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PMID:Isolation and chemical characterization of a melanoma-associated proteoglycan antigen. 661 28

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.
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PMID:A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage. 679 63

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.
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PMID:Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density. 711 9

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro. 753 59

Dermatan sulfate proteoglycans (DSPG) were extracted from intima-media of grossly normal aortic tissue of White Carneau pigeons and were purified by ion exchange chromatography on DEAE-Sephacel followed by size exclusion chromatography on Sepharose CL-4B. The major aortic DSPG had an average size of 310 kDa. The core protein resulting from treatment of the PG with chondroitinase ABC: (1) was found to be approximately 48 kDa by SDS-polyacrylamide gel electrophoresis; (2) was recognized by monoclonal antibody (Mab) 2-B-6 but not by Mab 3-B-3 on Western blots, indicating the presence of delta Di-4S and absence of delta Di-6S; (3) was glycosylated with Asn-linked oligosaccharides; (4) contained a high content of Asx, Glx and Leu, similar to that found for core proteins of this size from other tissues and species and (5) contained an N-terminal sequence (Asp-Glu-Gly-Xaa-Ala-Asp-Met-Pro-Pro-Xaa-Asp-Asp-Pro-Val- Ile-(ile)-Gly-Phe-), which was similar to sequences of DSPG core proteins previously described as 'decorin' and distinct from DSPG described as 'biglycan'. The results suggest that the major DSPG of aorta can be classified as a decorin molecule. The overall size of the DSPG in aorta was larger than decorin molecules described in non-arterial tissues of other species. Evidence is presented to conclude the larger size results from more than one dermatan sulfate-glycosaminoglycan chain.
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PMID:Structural properties and partial protein sequence analysis of the major dermatan sulfate proteoglycan of pigeon aorta. 845 55

Articular cartilage is both morphologically and biochemically heterogeneous. Its susceptibility to degenerative diseases such as arthritis and its limited repair capacity has made cartilage the focus of intense study; surprisingly, little is known of its development. Using a panel of specific antibodies, we have documented the temporal and spatial patterns of the small leucine-rich proteoglycans fibomodulin, decorin and biglycan in the developing knee cartilage of the marsupial South American opposum (Monodelphis domestica) from parturition to adulthood. The major proteoglycan of cartilage, aggrecan, can be substituted with a variety of isomers of chondroitin sulphate (CS) and keratan sulphate (KS) glycosaminoglycans. Consequently, we have used monoclonal antibodies to determine the distribution of the chondroitinase generated epitopes of CS isomers (delta di-6S and delta di-4S oligosaccharide 'stubs'). Other monoclonal antibodies (3B3[-], 7D4) were used to investigate temporal changes in the expression of specific sulphation patterns within native chondroitin sulphate chains in addition to keratan sulphate chains (5D4). We found the distributions of the small proteoglycans (PGs) to be highly dynamic during development. Both fibromodulin and biglycan appeared to specifically label early articular cartilage as opposed to epiphyseal or growth plate cartilage. All 3 small PGs become preferentially distributed to the upper half of the adult articular cartilage depth. Similarly, delta di-6S, delta di-4S oligosaccharide 'stubs', KS and epitope 7D4 were variably distributed during development but all were again preferentially located to the upper depth of the mature tissue. The epitope recognised by antibody 3B3[-] was extensively distributed in the neonate, but became more restricted to hypertrophic chondrocytes by day 19. It was not detected in the adult tissue. These data suggest that in Monodelphis, proteoglycans are preferentially synthesised and elaborated in the upper half of the tissue depth and contrasts with the patterns observed in eutherian mammals. The data also pose questions as to the functional significance of these molecules within the tissues and to the idea that global patterns of matrix components exist in mammalian articular cartilages.
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PMID:The development of articular cartilage: II. The spatial and temporal patterns of glycosaminoglycans and small leucine-rich proteoglycans. 918 84

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.
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PMID:Heparin cofactor II-dependent antithrombin activity of calcium spirulan. 887 66


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