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)

The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response.
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PMID:Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells. 309 24

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.
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PMID:Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia. 310 70

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.
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PMID:Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin. 621 15

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.
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PMID:Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta. 622 69

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca2+, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50-100 times faster at 22 degrees C than at 4 degrees C and was 5-10 times faster at 37 degrees C than at 22 degrees C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20-30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10-15 times faster in the absence of 1 mM Ca2+ in the buffer solution and was approximately 100 times faster in the presence of 0.1 M D-mannose as compared to 0.1 M D-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A2 and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation, Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2-4 h incubation at 4 degrees C was observed.
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PMID:Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells. 684 Nov 39

Colon wall from pig, stripped of most of the mucosal layer to leave material largely composed of muscle, basement membrane, and extracellular matrix, was subjected to procedures for isolation of glycosaminoglycans. A total ethanol precipitate from a papain digest was fractionated by selective ethanol precipitation in the presence of Ca2+. Glycosaminoglycan fractions, freed proteolytically from a high molecular weight glycoprotein component, were further purified by Sepharose CL-6B gel-filtration or DE-52 anion-exchange chromatography. Glycosaminoglycans were identified by chemical composition, 13C-NMR spectroscopy and response to chondroitinase and nitrous acid degradations. The content of glycosaminoglycan in the tissue is low (0.05% dry weight) being comprised of dermatan sulphate (38%), heparin (34%), heparan sulphate (18%) and chondroitin sulphates (10%) as a percentage of total glycosaminoglycan content. Hyaluronic acid and keratan sulphate have not been detected. The composition is generally typical of a high muscle content tissue.
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PMID:The glycosaminoglycans of pig colonic wall connective tissue. 684 74

Monoclonal antibodies have been used to study the presence and distribution of various components of the proteoglycan molecule in the intervertebral disc and cartilage endplate. Link protein, hyaluronic acid binding region, keratan sulphate and chondroitin 4- and 6-sulphate have been investigated in tissues from humans and other mammals. Exposure of the carbohydrate and protein epitopes was enhanced by chondroitinase and trypsin pretreatment respectively. The degree of immunoreactivity varied with location, being greater in the nucleus pulposus than the annulus fibrosus with least reactivity in the cartilage endplate. In addition, there was increased staining in the pericellular domains, particularly in adult tissues. Areas of ectopic calcification exhibited very different immunoreactivity, depending on the type of calcium salt present. Calcium hydroxyapatite deposits showed greater staining for 8A4 (link protein), while calcium pyrophosphate deposits demonstrated greater staining for 3B3(-), 7D4(-) and 3D5 than the surrounding non-calcified matrix. Staining for chondroitin sulphate isomer epitopes 3B3(-) and 7D4(-), indicative of modified chondroitin sulphate chains, was greater in human tissues of degenerate than non-degenerate appearance. This suggests that expression of these epitopes may be an indicator of disease and subsequent reparative procedures in intervertebral disc and cartilage endplate, similar to that seen in articular cartilage degeneration.
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PMID:Proteoglycan components of the intervertebral disc and cartilage endplate: an immunolocalization study of animal and human tissues. 751 84

A liposome-centered endogenous precipitation method was used to investigate the effect of ultrafilterable fragments from the enzymatic digestion of rat chondrosarcoma aggrecan on the formation of insoluble calcium phosphate salts in buffered solutions at pH 7.4 and 22 degrees C. Unlike the intact aggrecan and its major chondroitin sulfate and core protein components, disaccharide units from chondroitinase degradation of the aggrecan and small (< 3 kg/mol molecular weight) fragments from protease digestion of the core structure were found to be only weakly inhibitory toward mineral formation. Corresponding reductions in Ca(2+)-binding indicate that these fragments were unable to absorb to active sites on the apatite surface for long enough periods to significantly hinder crystal growth. The data suggest that controlled enzymatic breakdown of aggrecan may be one possible mechanism by which the calcification of growth plate cartilage is allowed to advance in vivo.
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PMID:Effect of ultrafilterable fragments from chondroitinase and protease-treated aggrecan on calcium phosphate precipitation in liposomal suspensions. 798 30

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 micrograms of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects (P < 0.005).
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PMID:Separate effects of urinary chondroitin sulphate and heparan sulphate on the crystallization of urinary calcium oxalate: differences between stone formers and normal control subjects. 814 91

Calpain I is a calcium-dependent cysteine proteinase that has been recently shown to degrade proteoglycan in vitro. The authors injected calpain I, which was purified from human red blood cells, into the intervertebral discs of rabbits. Roentgenograms showed disc space narrowing 1 week after the injection. Histologically, proteoglycan of the nucleus pulposus and anulus fibrosus decreased and notochordal cells in the nucleus pulposus almost disappeared. Biochemical data of the nucleus pulposus showed that the amounts of smaller proteoglycans increased 1 and 4 weeks after the injection. Eight weeks after the injection, histologic and biochemical data showed recovery compared with the data 1 week after injection. These findings show that calpain I is as potent an enzyme as chondroitinase ABC and has milder chemonucleolytic action than chymopapain. Regarding its possible clinical application, autogenous calpain I as purified from the patient's own red blood cells may have advantages over chymopapain and chondroitinase ABC in that it will prevent anaphylactic reaction.
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PMID:Chemonucleolysis with calpain I in rabbits. 843 17


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