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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)

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS-PAGE of the treated cells showed incorporation of label into a broad 97-121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97-121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5'-O-(3-thiotriphosphate). The 97-121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5'-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteolgycans.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins. 130 66

Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.
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PMID:The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A. 138 48

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.
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PMID:Components and proteolytic processing sites of arylsulfatase B from human placenta. 139 Sep 29

We have investigated the ability of glycosaminoglycans from embryonic chick brain (15 days old) to interact with basic fibroblast growth factor (bFGF). 35SO4 metabolically labeled glycosaminoglycans were purified and separated on DEAE-cellulose chromatography. Material which eluted between 0.20 and 0.35 M NaCl displaced the binding of [125I]bFGF to brain membrane. This activity was dose-dependent and on the basis to its heparinase sensitivity and chondroitinase insensitivity, has been attributed to heparan sulfate. CL-6B-Sepharose chromatography of this material revealed two glycosaminoglycans of molecular masses of about 15,000 and 65,000. Incubation with [125I]bFGF followed or not by heparinase and chondroitinase treatment of electrotransfert from SDS-PAGE revealed that both of these forms correspond to heparan sulfate chains and bind bFGF. In vitro, embryonic brain-derived heparan sulfate inhibited both bFGF induced [3H]thymidine incorporation in CCL39 cells and neurite outgrowth in PC12 cells. These results suggest that heparan sulfate play an important function in the control of the biological activity of bFGF during brain development.
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PMID:Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor. 139 71

The incorporation of [35S]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of 35S-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80% of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that 35S-labelled macromolecules isolated from the cell extract migrated with Kav. of 0.36, while corresponding material isolated from the medium migrated with Kav. of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise 35S-labelled proteins of various molecular masses. The 35S label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [35S]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [35S]sulphite from [35S]sulphate. The release of [35S]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [35S]sulphite reacted with these proteins to form a stable 35S-labelled conjugate.
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PMID:Synthesis of 35S-labelled macromolecules by polymorphonuclear neutrophils. Evidence for the production of [35S]sulphite which can modify both endogenous and exogenous proteins. 146 61

Proteoglycans synthesized by periodontal (gingival, periodontal ligament, dental follicle) fibroblasts were analysed by SDS/polyacrylamide and agarose gel electrophoresis after being labelled with radioactive sulphate. Medium, cell membrane and extracellular matrix fractions were analysed separately. Samples were treated with chondroitinase AC, chondroitinase ABC, heparitinase or a combination of chondroitinase ABC and heparitinase before electrophoretic separation of proteoglycans. Antibodies to versican and decorin were used to identify these molecules by Western immunoblots. For steady-state metabolic radiolabelling of fibroblasts, medium and cell membrane fractions contained about equal proportions of radiolabelled proteoglycans (about 43%), whereas less radioactivity (about 14%) was found in proteoglycans of the matrix fraction. Periodontal fibroblasts produce six major proteoglycans: versican, a high-molecular-mass chondroitin sulphate proteoglycan (CSPG); decorin, a dermatan sulphate proteoglycan (DSPG); a membrane-associated heparan sulphate proteoglycan (HSPG); two medium- or matrix-associated HSPGs; and a 91 kDa membrane-associated CSPG. Variation in decorin molecular size was observed in mass cultures of fibroblasts. Similar polydispersity in molecular size of decorin was seen in several clones established from one mass culture.
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PMID:A biochemical analysis of human periodontal tissue proteoglycans. 159 5

FN-C/H II is a heparin binding synthetic peptide from the C-terminal cell and heparin binding domain of fibronectin (FN) that mediates neuronal cell adhesion, spreading, and neurite outgrowth. Cellular interactions with FN-C/H II are inhibited by soluble heparin, suggesting that a cell-surface proteoglycan may mediate interactions with FN-C/H II (Haugen et al., 1990). To test this hypothesis further, heparan sulfate (HS) or chondroitin sulfate (CS) was removed from the cell surface by enzyme treatment. Heparitinase but not chondroitinase treatment of cells inhibited rat B104 neuroblastoma cell adhesion and spreading on FN-C/H II. Additionally, heparitinase treatment decreased the spreading of cells on the 33/66 kDa fragments containing the C-terminal heparin binding domain of FN. Furthermore, antibodies generated against a mouse melanoma HS proteoglycan (HSPG) inhibited B104 cell adhesion to FN-C/H II and the 33/66 kDa FN fragments. 35S-HSPG isolated from B104 cells directly bound to FN-C/H II both in solid phase assays and by affinity chromatography, but failed to bind to a control peptide from this region, CS1. The binding of 35S-HSPG was predominantly mediated by the HS and not the core protein of the HSPG. SDS-PAGE of iodinated HSPG demonstrated a single 78 kDa core protein following heparitinase digestion, which migrated at 51 kDa under nonreducing conditions. Anti-HSPG antibodies recognized the 78 kDa core protein by immunoblotting, and stained the surface of rat B104 neuroblastoma cells and cells of the primary neonatal rat nervous system. These results identify a cell-surface HSPG that likely mediates neuronal cell binding interactions with FN-C/H II.
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PMID:A cell-surface heparan sulfate proteoglycan mediates neural cell adhesion and spreading on a defined sequence from the C-terminal cell and heparin binding domain of fibronectin, FN-C/H II. 161 50

A simple and rapid procedure involving immunoadsorbent column chromatography has been developed for the isolation of lysosomal arylsulfatase B from human placenta. Using this method, we purified the enzyme over 20,000-fold with better recovery (16%) compared to that achieved by the conventional procedure. The enzyme appeared to be homogeneous and had an apparent molecular weight of 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified enzyme migrated as two bands with apparent molecular weights of 43,000 and 8,000 by reductive SDS-PAGE.
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PMID:Use of immunoadsorbent column chromatography for improved purification of arylsulfatase B from human placenta. 163 99

Colony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity-purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375- and 250-kDa with a smaller amount of a 100-kDa species. Analysis by gel filtration in 4 M guanidine HCI buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The three predominant CSF-1 species were shown to be an 80-kDa homodimer, an 80-kDa/50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80- and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase, and endo-alpha-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyl-terminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid-precipitable counts released from 35SO4(2-)-labeled L cells, has important implications for regulation by this growth factor.
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PMID:The predominant form of secreted colony stimulating factor-1 is a proteoglycan. 173 26

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.
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PMID:Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium. 174 72


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