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)

This paper describes a procedure that allows detection of endogenous heparin present in normal human plasma by polyacrylamide-gel electrophoresis (PAGE). Plasma was submitted to proteolysis: an antithrombin III-dependent and heparinase I-sensitive anticoagulant activity was demonstrated in the supernatant of the digest. The supernatant was submitted to sequential fractionation with increasing concentrations of ethanol (25%, 50%, 60% and 65%, by vol.). Fractions were analyzed by PAGE for both glycosaminoglycan (GAG) and protein content. GAGs were detected by gradient PAGE (24-30%). The fraction obtained by 60% ethanol precipitation contained heparinase I-sensitive GAG. We show that GAGs co-precipitate with proteins. The SDS-PAGE of the material resulting from proteolytic digestion and subsequent ethanol fractionation, revealed three major bands. These peptides co-precipitated with plasma GAGs, mainly with the fraction obtained by 60% ethanol. We discuss the possibility that circulating endogenous heparin interacts with such peptides.
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PMID:Detection of heparin-like glycosaminoglycans in normal human plasma by polyacrylamide-gel electrophoresis. 885 63

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.
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PMID:Novel purification and detailed characterization of perlecan isolated from the Engelbreth-Holm-Swarm tumor for use in an animal model of fibrillar A beta amyloid persistence in brain. 888 31

The origin of the heparan sulfate proteoglycan (PG), perlecan, in beta-amyloid protein (A beta)-containing amyloid deposits in Alzheimer's disease (AD) brain is not known. In the present investigation we used indirect immunofluorescence, SDS-PAGE, and Western blotting with a specific perlecan core protein antibody to identify possible cell candidates of perlecan production in both primary cell cultures and in a rat infusion model. Double and triple-labeled indirect immunofluorescence was performed on dissociated primary rat septal cultures using antibodies for specific identification of cell types and for perlecan core protein. In mixed cultures of both embryonic day 18 (containing neurons and glia) and postnatal day 2-3 (devoid of neurons), microglia identified by labeling with OX-42 or anti-ED1 were the only cell type also double labeled with an affinity-purified polyclonal antibody against perlecan core protein. Similar immunolabeling of microglia with the anti-perlecan antibody was also observed in purified cultures of post-natal rat microglia. Analyses of PGs from cultured postnatal rat microglia by Western blotting using a polyclonal antibody against perlecan core protein revealed an approximately 400 kDa band in cell layer, which was intensified following heparitinase/heparinase digestion, suggestive of perlecan core protein. Other lower Mr bands were also found implicating either degradation of the 400 kDa core protein or the presence of separate and distinct gene products immunologically related to perlecan. Reverse transcription followed by polymerase chain reaction using human perlecan domain I specific primers demonstrated perlecan mRNA in cultured human microglia derived from postmortem normal aged and AD brain. Following a 1-week continuous infusion of A beta (1-40) into rodent hippocampus, immunoperoxidase immunocytochemistry and double-labeled immunofluorescent studies revealed perlecan accumulation primarily localized to microglia/macrophages within the A beta infusion site. These studies have identified microglia/macrophages as one potential source of perlecan (or a perlecan-related macromolecule) which may be important for the ongoing accumulation of both perlecan and A beta in the amyloid deposits of AD.
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PMID:Localization of perlecan (or a perlecan-related macromolecule) to isolated microglia in vitro and to microglia/macrophages following infusion of beta-amyloid protein into rodent hippocampus. 933 37

Sixteen heparinase-producing isolates, related to Sphingobacterium heparinum, were grouped into three major clusters by SDS-PAGE and DNA-rRNA hybridizations. Based on a polyphasic approach, it was shown that isolates of two of these clusters and S. heparinum species belong to a new genus for which the name Pedobacter is proposed. The genus consists of Pedobacter heparinus comb. nov. (formerly Sphingobacterium heparinum), which is the type species, Pedobacter piscium comb. nov. (formerly Sphingobacterium piscium), Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. and four as-yet-unnamed DNA hybridization groups. All the previously named taxa can be discriminated by phenotypic features, but have strong overall similarities with representatives of the genus Sphingobacterium and the misclassified species [Flexibacter] canadensis. All these organisms constitute a separate rRNA branch in rRNA superfamily V for which the family Sphingobacteriaceae fam. nov. is proposed.
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PMID:Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. proposal of the family Sphingobacteriaceae fam. nov. 954 86

Two novel acharan sulfate lyases (ASL1 and ASL2: no EC number) have been purified from Bacteroides stercoris HJ-15 which was isolated from human intestinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzymes were purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, carboxymethyl-Sephadex C-50, hydroxyapatite and HiTrap SP Sephadex C-25 column chromatography with the final specific activity of 50.5 and 76.7 micromol.min-1.mg-1, respectively. Both acharan sulfate lyases are single subunits of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed optimal activity at pH 7.2 and 45 degrees C. ASL1 activity was inhibited by Cu2+, Ni2+ and Co2+, but ASL2 activity was inhibited by Cu2+, Ni2+and Pb2. Both enzymes were slightly inhibited by some agents that modify histidine and cysteine residues, but activated by reducing agents such as DL-dithiothreitol and 2-mercaptoethanol. Both purified bacteroidal acharan sulfate lyases acted to the greatest extent on acharan sulfate, and to a lesser extents on heparan sulfate and heparin. They did not act on de-O-sulfated acharan sulfate. These findings suggest that the biochemical properties of these purified acharan sulfate lyases are different from those of the previously purified heparin lyases, but these enzymes belong to heparinase II.
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PMID:Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15. 1132 84

The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4 degrees C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP(5) and that the N protein bound to heparin as a homodimer. GP(3), which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP(5) complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.
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PMID:Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. 1193 97

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.
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PMID:Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans. 1209 42

The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.
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PMID:Cloning, sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate. 1240 Jun 86

Salt-active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with GAG degrading enzymes. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, diethylaminoethyl (DEAE)-cellulose, CM-Sephadex C-50, HA ultrogel and phosphocellulose column chromatography with the final specific activity of 81.33 micro mol x min-1 x mg-1. The purified salt-active acharan sulfate lyase was activated to 5.3-fold by salts (KCl and NaCl). The molecular weight of salt-active acharan sulfate lyase was 94 kDa by SDS/PAGE and gel filtration. The salt-active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 degrees C. Salt-active acharan sulfate lyase activity was potently inhibited by Cu2+, Ni2+ and Zn2+. This enzyme was inhibited by some agents, butanediol and p-chloromercuric sulfonic acid, which modify arginine and cysteine residues. The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate, to a lesser extent on heparan sulfate and heparin. The biochemical properties of the purified salt-active acharan sulfate lyase are different from those of the previously purified heparin lyases. However, these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II.
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PMID:Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15. 1286 91

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.
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PMID:Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15. 1560 27


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