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)

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).
Biochem J 1991 Sep 15
PMID:A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains alpha (2-3)-linked N-acetylneuraminic acid. 191 Mar 36

Ultrastructural localization of proteoglycans (PGs) in 1-week- to 2-year-old scar was determined by staining with cuprolinic blue dye (CBD) after specific enzymatic digestion of keratan sulfate (KS) glycosaminoglycans (GAGs) or chondroitin sulfate glycosaminoglycans (CSs). High critical electrolyte conditions were maintained for CBD-staining, specific for high-sulfated GAGs. Although KS was detected in the 1-week-old wound, no CBD-stained KS was seen in the anterior stroma adjacent to the wound. The CS was present throughout the 1-week-old wound and adjacent stroma, and PGs were biosynthetically 35SO4-labeled in normal stroma. Subsequently, radioactivity from labeled PGs in normal stroma adjacent to the wound moved into scar tissue during healing. Marked sensitivity of PGs to Chondroitinase ABC indicated an abundance of CS in 2-week-old scars. Punctate CBD-staining and immunohistochemical evidence suggested chemically altered KS is present in the 2-week-old anterior scar. The pattern of CBD-staining in 1- and 2-week scars, after chondroitinase treatment, suggested KS in the younger scar is similar to adult high-sulfated GAG, whereas KS in the 2-week scar contains primarily newly synthesized low-sulfated KS. The latter is consistent with previous immunochemical and biochemical analyses. Cytochemical and immunohistochemical evidence indicated that KS is not present in the 2-week-old posterior scar. By the week 8 of healing, CBD-stained KS was present throughout most of the scar, except along the posterior margin, consistent with earlier stages of healing. The CBD-stained structures in the first 8 weeks of healing were reminiscent of stained GAGs in normal developing cornea. This fetal-like CBD-staining pattern seen in scar, however, changed to that of the normal adult by the 2nd year of healing. The significance of these observations relate to our contention that healing adult cornea recapitulates some ontogenetic events of the normal cornea, and that the nonuniform distribution and chemical properties of GAGs in scar tissue are a function of the movement of existing proteoglycans and de novo synthesis of altered macromolecules.
Invest Ophthalmol Vis Sci 1990 Sep
PMID:Morphologic analyses of proteoglycans in rabbit corneal scars. 212 Jan 45

Previous studies on rabbit thrombomodulin (TM) revealed that certain anticoagulant activities expressed by TM depend on the presence of an acidic domain tentatively identified as a sulfated galactosaminoglycan (Bourin, M.-C., Ohlin, A.-K., Lane, D., Stenflo, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 8044-8052). The glycan was released by alkaline beta-elimination, isolated by ion-exchange chromatography, and radiolabeled by partial N-deacetylation (hydrazinolysis) followed by re-N-[3H]acetylation. The labeled product behaved like standard chondroitin sulfate on ion-exchange chromatography, exhibited a Mr of 10-12 x 10(3) on gel chromatography, and was susceptible to degradation by chondroitinase and testicular hyaluronidase. The major labeled degradation products following digestion of the glycosaminoglycan with chondroitinase were identified, depending on the incubation conditions, either as 4/6-mono-O-sulfated, 4,5-unsaturated disaccharides (delta HexA-GalNAc(S] and N-acetylgalactosamine 4,6-di-O-sulfate (GalcNAc (diS], the latter component accounting for approximately 25% of the total label, or as a major fraction of labeled trisaccharide, with the predominant structure GalNAc(diS)-GlcA-GalNAc(diS). The terminal GalNAc(diS) unit (not substituted at C3) was shown to be more susceptible to N-deacetylation during hydrazinolysis than were the internal GalNAc units (substituted at C3), and thus was more extensively labeled, resulting in over-representation of this unit. It is concluded that rabbit TM is a chondroitin sulfate proteoglycan, which carries a single glycan side chain characterized by an unusual accumulation of sulfate groups at the nonreducing terminus. Metabolically 35S-labeled TM was isolated from cultured rabbit heart endothelial cells and characterized as a chondroitin sulfate proteoglycan which accounted for 1-2% of the total 35S-labeled cell-associated macromolecules. The isolated chondroitin sulfate showed weaker antithrombin-dependent anticoagulant activity, on a molar basis, than the intact TM proteoglycan. The anticoagulant action of TM thus depends on a unique form of functional collaboration between the different constituents of a glycoconjugate.
J Biol Chem 1990 Sep 15
PMID:Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. 216 13

Thrombomodulin (TM), a major anticoagulant protein at the vessel wall, serves as a potent cofactor for the activation of Protein C by thrombin. Previous work has indicated that (rabbit) TM is a proteoglycan that contains a single polysaccharide chain, tentatively identified as a sulphated galactosaminoglycan, and furthermore suggested that this component may be functionally related to additional anticoagulant activities expressed by the TM molecule [Bourin, Ohlin, Lane, Stenflo & Lindahl (1988) J. Biol. Chem. 263, 8044-8052]. Results of the present study establish that (enzymic) removal of the polysaccharide chain abolishes the inhibitory effect of TM on thrombin-induced fibrinogen clotting as well as the promoting effect of TM on the inactivation of thrombin by antithrombin, but does not affect the ability of TM to serve as a cofactor in the activation of Protein C. Studies of yet another biological activity of rabbit TM, namely the ability to prevent the activation of Factor V by thrombin [Esmon, Esmon & Harris (1982) J. Biol. Chem. 257, 7944-7947], confirmed that TM markedly delays the conversion of the native 330 kDa Factor V precursor into polypeptide intermediates, and further into the 96 kDa heavy chain and 71-74 kDa light-chain components of activated Factor Va. In contrast, the activation kinetics of a similar sample of Factor V incubated with thrombin in the presence of chondroitinase ABC-digested TM did not differ from that observed in the absence of TM. It is concluded that the inhibitory effect of TM on Factor V activation also depends on the presence of the polysaccharide component on the TM molecule.
Biochem J 1990 Sep 01
PMID:Functional role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Effects on inactivation of thrombin by antithrombin, cleavage of fibrinogen by thrombin and thrombin-catalysed activation of factor V. 216 42

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.
Biochem Biophys Res Commun 1990 Sep 14
PMID:Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. 216 32

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.
Arch Biochem Biophys 1990 Sep
PMID:Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A. 239 98

Corneal buttons were obtained from patients with types 1 and 2 macular corneal dystrophy (MCD) and from control patients with Fuchs' dystrophy or keratoconus. Buttons were incubated for 20 h in the presence of [3H]glucosamine or [2-3H]mannose. Radiolabeled proteoglycans and lactosaminoglycan-glycoproteins (L-GPs) were purified using chromatography on Q-Sepharose, Superose 6, and octyl-Sepharose. They were identified using chondroitinase ABC, keratanase or endo-beta-galactosidase digestion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Superose 6 chromatography. This study confirms previous reports that type 1 MCD corneas synthesize a normal dermatan sulfate-proteoglycan (DS-PG) and an abnormal keratan sulfate-proteoglycan (KS-PG). The data indicate that typ 1 MCD corneas synthesize L-GP instead of KS-PG. This L-GP has a core protein of similar hydrophobicity (elution from octyl-Sepharose) and nearly similar mass (42 kDa) as the core protein of the KS-PG. It has identical glycoconjugates as those of the KS-PG except that they lack sulfate. Thus, type 1 MCD fails to synthesize keratan sulfate as a result of a defect in a sulfotransferase specific for sulfating lactosaminoglycans. Further, proteoglycans synthesized by a cornea from a patient with type 2 MCD were studied. This cornea synthesized a normal ratio of KS-PG to DS-PG although net synthesis of proteoglycans was approximately 30% below normal. The KS-PG appeared normal whereas the DS-PG had dermatan sulfate chains that were approximately 40% shorter than normal.
J Biol Chem 1990 Sep 15
PMID:Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy. 239 54

Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
J Biol Chem 1985 Sep 15
PMID:Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells. 241 30

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.
J Biol Chem 1988 Sep 25
PMID:Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor. 245 54

To gain insight into the cellular and molecular mechanisms underlying cell interactions in the early postnatal mouse cerebellum, Ca2+-dependent and -independent aggregation mechanisms were characterized using single cell suspensions under conditions that allow discrimination between the two mechanisms. When cerebellar cells were derived from newborn to 10-day-old mouse cerebellum, both mechanisms were active and showed no major change in activity during this time period. Mg2+ could not replace Ca2+ in the Ca2+-dependent mechanism. In contrast to the Ca2+-independent mechanisms, the Ca2+-dependent mechanism was inactive at low temperatures, suggesting a necessity for molecular rearrangement within the surface membrane during aggregation. Neuraminidase, chondroitinase, heparinase or hyaluronidase treatment of cells did not influence the aggregation of cells under Ca2+-dependent and -independent conditions. Chondroitin sulfate inhibited and hyaluronic acid stimulated the Ca2+-dependent mechanism, whereas chondroitin sulfate only slightly and hyaluronic acid strongly inhibited the Ca2+-independent one. Dextran sulfate slightly inhibited both mechanisms, whereas heparin and fucoidan, a complex sulfated carbohydrate, did not influence cell aggregation, while they strongly inhibited attachment of cells to laminin. The polycation poly-L-lysine slightly stimulated the Ca2+-independent mechanism, but inhibited the Ca2+-dependent one. Interestingly, chondroitin sulfate and hyaluronic acid strongly stimulated cell aggregation under conditions where both mechanisms were almost destroyed or inactive. Dextran sulfate showed only a small effect under these conditions. These observations indicate that different molecular mechanisms are active in cell-cell versus cell-extracellular matrix interactions and suggest a hitherto unknown complexity in molecular mechanisms during early postnatal cerebellar development.
Brain Res 1988 Sep 01
PMID:Characterization of Ca2+-dependent and -independent aggregation mechanisms among mouse cerebellar cells. 246 13


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