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:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.
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PMID:Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin. 772 74

The binding of albumin to the glomerular capillary wall was studied using albumin-gold in perfused kidneys, the interaction of [3H]albumin with isolated glomeruli at 37 degrees C and 4 degrees C and the interaction at [3H]albumin with purified basement membrane. The albumin-gold was found to bind predominantly to the basement membrane and this interaction could be dissociated with high concentrations of albumin. There was binding of albumin to isolated rat glomeruli which exhibited temperature dependence. Glomeruli exhibited a binding site at both 37 degrees C and 4 degrees C with an association constant in the range of 1 to 3 x 10(4) M-1 that bound 7 x 10(13) molecules/glomerulus. At 37 degrees C, however, there was anomalous Scatchard binding behaviour at relatively higher concentrations of albumin (30 to 50 mg/ml) which could be due to either glomerular cell uptake or the appearance of multiple binding sites or both. The binding of albumin to isolated glomeruli and the glomerular albumin levels in isolated kidney perfusion could largely be accounted for by the binding of albumin to the glomerular basement membrane. The albumin binding to glomeruli at 37 degrees C was enhanced by Pronase digestion and heparinase digestion, but remained unchanged following trypsin treatment or neuraminidase treatment. Similarly, albumin was shown to bind to purified basement membrane preparations. This binding was also enhanced (approximately 80 times) by heparinase digestion but remained unchanged after digestion with chondroitinase ABC or hyaluronidase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Albumin interaction with the glomerular capillary wall in vitro. 778

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.
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PMID:Analysis of the lipoprotein binding site of rat liver membranes. 781 47

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
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PMID:Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin. 787 96

The action pattern of polysaccharide lyases on glycosaminoglycan substrates was examined using viscosimetric measurements and gradient polyacrylamide gel electrophoresis (PAGE). Heparin lyase I (heparinase, EC 4.2.2.7) and heparin lyase II (no EC number) both acted on heparin in a random endolytic fashion. Heparin lyase II showed an ideal endolytic action pattern on heparan sulphate, while heparin lyase I decreased the molecular weight of heparan sulphate more slowly. Heparin lyase III (heparitinase, EC 4.2.2.8) acted endolytically only on heparan sulphate and did not cleave heparin. Chondroitin ABC lyase (chondroitinase ABC, EC 4.2.2.4) from Proteus vulgaris acted endolytically on chondroitin-6-sulphate (chondroitin sulphate C) and dermatan sulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate (chondroitin sulphate A) at a reduced rate, decreasing its molecular weight much more slowly. Two chondroitin AC lyases (chondroitinase AC, both EC 4.2.2.5) were examined towards chondroitin-4- and -6-sulphates. The exolytic action of chondroitin AC lyase A from Arthrobacter aurescens on both chondroitin-4- and -6-sulphates was demonstrated viscosimetrically and confirmed using both gradient PAGE and gel permeation chromatography. Chondroitin AC lyase F from Flavobacterium heparinum (Cytophagia heparinia) acted endolytically on the same substrates. Chondroitin B lyase (chondroitinase B, no EC number) from F.heparinum acted endolytically on dermatan sulphate giving a nearly identical action pattern as observed for chondroitin ABC lyase acting on dermatan sulphate.
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PMID:Action pattern of polysaccharide lyases on glycosaminoglycans. 794 54

Mesoderm forms in the vertebrate embryo as a result of inductive interactions involving secreted growth factors and cell surface molecules. Proteoglycans have recently been implicated in the control of cell adhesion, migration and growth factor responsiveness. We have found that removal of glycosaminoglycan chains of proteoglycans from Xenopus ectodermal explants by heparinase, but not by chondroitinase, results in inhibition of elongation and mesodermal differentiation in response to signaling factors: activin, FGF and Wnt. Heparinase treatment differentially affected expression of early general and region-specific mesodermal markers, suggesting that mesodermal cell fates become specified in the early embryo via at least two signaling pathways which differ in their requirements for heparan sulfate proteoglycans. Addition of soluble heparan sulfate restored activin-mediated induction of muscle-specific actin gene in heparinase-treated explants. Finally, heparinase inhibited autonomous morphogenetic movements and mesodermal, but not neural, differentiation in dorsal marginal zone explants, which normally give rise to mesoderm in the embryo. These results directly demonstrate that heparan sulfate proteoglycans participate in gastrulation and mesoderm formation in the early embryo.
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PMID:Heparan sulfate proteoglycans are required for mesoderm formation in Xenopus embryos. 795 42

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.
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PMID:Preparation and characterization of deuterium-labeled glycosaminoglycans. 799 88

Previous studies have identified glycosaminoglycans in gingival crevicular fluid (GCF) associated with a variety of clinical conditions, notably those involving bone resorptive activity. GCF was here collected from around teeth undergoing active orthodontic movement. Proteoglycan metabolites were purified from GCF by anion-exchange chromatography using fast performance liquid chromatography. Sulphated glycosaminoglycan was associated with the most highly anionic protein fractions IV, V and VI, and biochemical analysis was restricted to these fractions. Analysis included glycosaminoglycan content by cellulose acetate electrophoresis, molecular size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and amino acid analyses. Fraction IV contained hyaluronan (18.7%) and chondroitin sulphate (10.9%), fraction V heparan sulphate (29.5%) and chondroitin sulphate (19.6%) and fraction VI chondroitin sulphate only (21.3%). SDS-PAGE revealed two Coomassie blue bands in fraction V of 72 and 60 kDa and two further bands in fraction VI of 71 and 56 kDa. These proteoglycans appeared resistant to digestion by chondroitinase ABC or heparinase III, although the glycosaminoglycan chains underwent degradation after protein-core removal. The molecular mass and amino acid composition of the chondroitin sulphate proteoglycan fractions showed a close similarity to those of human alveolar bone proteoglycan. The presence of heparan sulphate proteoglycan in GCF in association with orthodontic movement is in accord with previous reports. The findings support the view that proteoglycans in GCF are 'biomarkers', notably those associated with active resorption of alveolar bone.
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PMID:Characterization of proteoglycan metabolites in human gingival crevicular fluid during orthodontic tooth movement. 806 Feb 58

Borrelia burgdorferi adhere to mammalian cells in vitro but neither the ligand(s) nor the receptor(s) has (have) been clearly established. Using an in vitro attachment-inhibition assay, a B. burgdorferi attachment mechanism has been identified. Heparin, heparan sulfate, and dermatan sulfate reduced the attachment of virulent B. burgdorferi strain 297 to HeLa cells by approximately 60%. In addition, virulent, but not avirulent, B. burgdorferi strains B31, N40, and HB19 demonstrated heparin attachment-inhibition. Attachment to Chinese hamster ovary cells deficient in heparan sulfate proteoglycans was reduced by 68% compared to attachment to wild-type cells and was identical to attachment at maximum heparin inhibition to the wild-type cells. Pretreatment of HeLa cell monolayers with heparitinase, heparinase, and chondroitinase ABC, but not with chondroitinase AC, reduced borrelial attachment by approximately 50%. A moderately high affinity, low copy number, promiscuous B. burgdorferi glycosaminoglycan receptor was demonstrated by equilibrium binding studies. A 39-kD polypeptide, purified by heparin affinity chromatography from Triton X-100 extracts derived from virulent borrelia, was a candidate for this receptor. These studies indicate that one mode of B. burgdorferi attachment to eukaryotic cells is mediated by a borrelial glycosaminoglycan receptor attaching to surface-exposed proteoglycans on mammalian cells.
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PMID:Borrelia burgdorferi bind to epithelial cell proteoglycans. 811 83

Recent studies have demonstrated that basic fibroblast growth factor (bFGF) plays a key role in the terminal differentiation of growth plate chondrocytes during endochondral ossification. We therefore examined the binding of [125I]bFGF to an extract of extracellular matrix (ECM) of rat growth plate. Using a solid phase binding assay, binding of [125I]bFGF to ECM was demonstrated to be specific and saturable at 0.5-1 microgram/ml of bFGF. Scatchard analysis demonstrated the presence of a single class of binding sites with an apparent Kd of 14 nM. The binding of [125I]bFGF to rat growth plate ECM was inhibited by the addition of heparin, heparan sulfate, and dermatan sulfate. The binding was reversible as these glycosaminoglycans were also effective at displacement of bound [125I]bFGF from rat growth plate ECM. Chondroitin 4- or 6-sulfate had no effect on either binding or displacement when added at the same concentrations. Preincubation of the rat growth plate ECM with heparinase or heparitinase resulted in a reduction of the binding of [125I]bFGF to the ECM. Furthermore, these enzymes were able to significantly displace bound growth factor. In contrast, chondroitinase ABC or AC failed to displace bound [125I]bFGF from the ECM. Similar results were obtained when matrix derived from rat growth plate tissue was used for the binding studies. The results demonstrate that bFGF binds to a heparan sulfate in matrix produced by rat growth plate chondrocytes and matrix extracted from rat growth plate and suggest that this glycosaminoglycan may serve as a storage depot for this growth factor during endochondral ossification.
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PMID:Basic fibroblast growth factor binds to heparan sulfate in the extracellular matrix of rat growth plate chondrocytes. 816 Dec 3


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