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)

Proteoglycans have been isolated from a high speed supernatant fraction of a mouse mastocytoma by procedures which should minimize alteration of the native protein-polysaccharide molecule. The methods used include in vivo labeling proteoglycans with 35S-sulfate, 3H-leucine and 3H-lysine, centrifugation of the tumor homogenate at 105,000 g, cetylpyridinium fractionation of the supernatant, and further purification of some of the fractions obtained by DEAE-cellulose column chromatography, gel filtration on Sepharose 4B and cellulose acetate electrophoresis. Two major sulfated proteoglycans were obtained, one containing keratan sulfate-like material (KSP-S), the other a heparin-like polymer (HP-S). The presence in HP-S of a compound similar to heparin was confirmed by its digestibility with flavobacterium heparinase. HP-S contained about 4 per cent protein. Glycine was the predominant amino acid, and serine did not appear to be involved in the peptide-carbohydrate linkage. The proteoglycan present in HP-S appeared to be homogeneous when examined using cellulose acetate electrophoresis. KSP-S was found to contain sialic acid and its protein content was significantly higher than that of HP-S. Glutamic and aspartic acids were the most abundant amino acids in KSP-S.
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PMID:Proteoglycans of soluble fraction of mouse mastocytoma. 12 69

Heparinase was isolated from a transplantable mouse mastocytoma, by salt extraction of a particulate fraction sedimenting at 20,000 times g, followed by precipitation from saturated ammonium sulfate. By use of gel chromatography through Sepharose 4B, the enzyme was shown to degrade macromolecular. 35S-labeled, mastocytomal heparin (K-av about 0.25) to products similar in size to commercial heparin (K-av about 0.85), apparently by nonrandom cleavage of a limited number of glycosidic linkages per molecule. Prolonged incubation times (up to 5 days, with repeated addition of enzyme) did not result in further degradation of the product. No significant depolymerizing activity was observed with any other glycosaminoglycan tested, including chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, and commercial heparin. The pH optimum for degradation of macromolecular heparin was around pH 5. The nature of the linkage cleaved by the heparinase was investigated by reduction of unlabeled polysaccharide degradation products with sodium [3H]borohydride. The degraded chains (but not the macromolecular substrate) incorporated significant amounts of tritium. An essentially monodisperse fraction of the labeled, degraded heparin was subjected to meniscus depletion sedimentation equilibrium ultracentrifugation, indicating a molecular weight of 14,500. By relating the molecular weight to the specific activity of the preparation, the amount of reducible groups was calculated to be approximately one per molecule. The 3H-labeled heparin was degraded to monosaccharides by a combination of acid hydrolysis and cleavage due to deamination with nitrous acid. Analysis of the degradation products, by paper electrophoresis and paper chromatography, showed a major radioactive component which behaved like L-gulonic acid. Since [3H]gulonic acid would be the expected reduction product of a polysaccharide molecule, containing a glucuronic acid residue in terminal position, these results tentatively suggest that the heparinase is an endoglucuronidase. By direct deaminative cleavage (no hydrolysis) of the 3H-labeled heparin, the glucosamine unit in penultimate position (i.e. adjacent to the [3H]gulonic acid residue) was shown to be 52% N-sulfated and 48% N-acetylated. As only 14% of the glucosamine was N-acetylated in the macromolecular heparin substrate, it is suggested that cleavage of this polysaccharide, by the heparinase, occurs in regions more abundant in N-acetylated glucosamine residues than other portions of the molecule. The possibility that formation and degradation of macromolecular heparin occurs also in mammalian species other than rodents in discussed.
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PMID:Cleavage of macromolecular heparin by an enzyme from mouse mastocytoma. 80 78

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.
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PMID:Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate. 757 59

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.
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PMID:Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction: target and inhibitor saccharides. 774 62

Contact activation occurs when plasma comes in contact with negatively charged manmade surfaces but no substance that initiates contact activation in vivo has been identified. We have isolated a mast cell heparin proteoglycan (MC-HepPG) from a Furth mouse mastocytoma-derived cell line that is analogous to human tissue-type mast cell HepPG. This material and other glycosaminoglycans (GAGs) were tested for their ability to accelerate the reciprocal activation of factor XII and prekallikrein and the autoactivation of factor XII. Quantitative analysis showed the MC-HepPG to be as active as dextran sulfate on a weight basis; hog intestine heparin, dermatan sulfate, keratan polysulfate and chondroitin sulfate C were less active, other sulfated polysaccharides were essentially inactive. Incubation of MC-HepPG in 1:4 diluted plasma resulted in complete cleavage of high molecular weight kininogen in a factor XII-dependent reaction. All of the MC-HepPG dependent reactions described above were inhibited by preincubation of MC-HepPG with heparinase I and II but not by pretreatment with heparitinase, chondroitinase ABC or the serine protease inhibitor aPMSF thus indicating that heparin proteoglycan is indeed acting as an initiating 'surface'. We analysed the proteoglycan preparation by HPLC gel filtration. Fractions spanning a molecular weight range of > 400000-8000 were active initiators. Comparison of the chromatograms obtained before and after cleavage of GAG side chains from the protein core suggested that dissociated GAGs in the MW range 69000-17000 are the most active species rather than the complete proteoglycan. MC-HepPG GAGs therefore represent a physiologic macromolecule with activity comparable to non-physiological surfaces in a purified system and with the capability to induce activation of the contact system in diluted plasma. Its ability to promote kinin generation links cellular and humoral inflammatory responses in the perivasculature and provides a possible explanation for the elevated kinin levels observed after allergen exposure.
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PMID:Mast cell derived heparin activates the contact system: a link to kinin generation in allergic reactions. 920 86

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [(35)S]sulphate (and in some cases [6-(3)H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [(3)H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.
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PMID:Biosynthesis of glycosaminoglycans by cultured mastocytoma cells. 1674 5