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:3.1.6.12 (chondroitinase)
2,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The basic subunit of cartilage proteoglycan consists of multiple glycosaminoglycan chains covalently attached to a core protein. It is unclear as to whether there is a single core protein or multiple different core proteins, since previous studies using either chondroitinase or testicular hyaluronidase to enzymatically remove chondroitin sulfate side chains from the proteoglycan subunit have yielded conflicting results. In the present study, a chondroitinase-produced core protein preparation isolated as a single peak on Sepharose gel chromatography was found to contain at least two immunologically distinct components. Hyaluronidase-produced core protein from the same proteoglycan subunit fraction was found to contain multiple components nearly all of which were smaller than the components in the chondroitinase digest. A possible explanation of these findings is that they resulted from proteolytic degradation of the core protein in the course of the enzymatic removal of its chondroitin sulfate. The presence of small amounts of protease contaminants in several commercial chondroitinase and hyaluronidase preparations was detected by an extremely sensitive radioassay. Until proteases can be rigorously excluded from enzyme preparations used to degrade the proteoglycan subunit, it will not be possible to determine whether it consists of a single or several different core proteins.
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PMID:A comparison of bovine nasal cartilage proteoglycan core protein produced by chondroitinase and hyaluronidase: the possible role of protease contaminants. 14 80

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. The findings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific for heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent major components of these sites since the latter are not affected by digestion with neuraminidase and other glycosaminoglycan-specific enzymes. Identical findings were obtained on basement membranes in other locations (Bowman's capsule, tubule epithelium, and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.
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PMID:Presence of heparan sulfate in the glomerular basement membrane. 15 19

Glycosaminoglycans were isolated from purified fractions of glomerular basement membranes and partially characterized by chemical analysis and cellulose acetate electrophoresis. Basement membranes were prepared by detergent treatment of rat glomeruli and subjected to digestion with papain and Pronase. Glycosaminoglycans were isolated from the digests by precipitation with cetyl pyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated glycosaminoglycan fraction revealed the presence of one major and one minor spot. The major spot was identified as heparan sulfate because it comigrated with the heparan sulfate standard and was sensitive to heparinase and to nitrous acid oxidation but insensitive to chondroitinase ABC and to testicular or leech hyaluronidase. The minor spot was tentatively identified as hyaluronic acid based on its migratory behavior and sensitivity to leech and testicular hyaluronidase. The chemical composition of the isolated glycosaminoglycan was typical of that of heparan sulfate (high carbazole/orcinol ratio, high sulfate content, absence of galactosamine). The data support and confirm the cytochemical data obtained previously [Kanwar, Y. S. & Farquhar, M. G. (1979) Proc. Natl. Acad. Sci. USA 76, 1303-1307] demonstrating that heparan sulfate is the only sulfated glycosaminoglycan detectable in the glomerular basement membrane. The present results suggest that in addition to sulfated glycosaminoglycan some nonsulfated glycosaminoglycan (hyaluronic acid) may also be present in the glomerular basement membrane.
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PMID:Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. 15 57

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.
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PMID:Collagenolytic activity of some marine bacteria. 16 14

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.
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PMID:Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments. 22 Sep 58

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.
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PMID:Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells. 37 19

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.
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PMID:Glycosaminoglycans of normal and malignant cultured human mammary cells. 42 76

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.
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PMID:Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B. 42 37

Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.
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PMID:Binding of soluble type I collagen molecules to the fibroblast plasma membrane. 45 36

This study demonstrates that the dorsal ectoderm of the stage 14 chick embryo synthesizes hyaluronic acid. About 49 to 52% of the H3 glucosamine-labeled glycosaminoglycan that is synthesized by explanted ectoderm can be identified as hyaluronic acid on the basis of its susceptibility to Streptomyces hyaluronidase or isolation of chondroitinase ABC digestion products. In addition, autoradiographic evidence shows that the ectoderm, unlike adjacent tissues like epithelial somites or neural tube, incorporates glucosamine into hyaluronidase-sensitive material which becomes largely extracellular and localized in the subectodermal cell-free space. Ultrastructural evidence shows that there is a fine fibrillar matrix between the ectodermal cells and in the subectodermal spaces when tannic acid is included in the primary fixative. This material resembles authentic hyaluronate, similarly fixed, and is absent when tannic acid is omitted from the fixative or when embryos have been previously treated in ovo with Streptomyces hyaluronidase. The concomitant reduction in the intercellular and subectodermal cell-free spaces after in ovo treatment with Streptomyces hyaluronidase supports the hypothesis that the dorsal ectoderm plays a morphogenetic role by contributing hyaluronate to the forming extracellular spaces. It is proposed that ectodermally derived hyaluronate might influence the morphogenesis of subjacent tissues such as the dermatome and neural crest.
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PMID:The synthesis of hyaluronic acid by ectoderm during early organogenesis in the chick embryo. 47 12


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