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)

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis.
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PMID:Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo. 804 Mar 12

Hepatocyte growth factor (HGF) purified from human placenta was specifically bound to a component in ECM secreted from lung fibroblasts, hepatocytes, and nonparenchymal liver cells. HGF was also bound to a component in basement membrane matrigel. The rate of DNA synthesis in hepatocytes cultured on the HGF-bound ECM was 4-7 times that of control hepatocytes. When ECM-coated dishes were pretreated with heparinase and heparitinase prior to binding of HGF, the stimulation decreased remarkably. A considerable decrease in the stimulation was observed following the washing of HGF-bound ECM with 1 M NaCl. We propose, based on these observations, that HGF bound to the ECM component present in Disse's space functions in regeneration following hepatic injury.
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PMID:Stimulation of DNA synthesis in hepatocytes by hepatocyte growth factor bound to extracellular matrix. 846 99

Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.
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PMID:Cloning and expression of heparinase I gene from Flavobacterium heparinum. 847 14

We have investigated the interaction of basic fibroblast growth factor (bFGF) with its receptors and heparan sulfate proteoglycans (HSPG). It has been suggested that in the absence of HSPG, cells are not able to bind bFGF or respond to treatment with bFGF. In our studies, Balb/c3T3 fibroblasts were treated with 50 mM sodium chlorate to completely inhibit (99%) sulfation of proteoglycans. We found that bFGF was able to bind, be internalized, and stimulate DNA synthesis in the absence of HSPG in a dose-dependent manner. bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity and no change in receptor number. To determine if this decreased affinity bFGF-receptor interaction is functional, we quantitatively analyzed bFGF internalization and stimulation of DNA synthesis in control and chlorate-treated cells. Endocytotic rate constants (ke) for chlorate-treated and control cells were ke = 0. 078 +/- 0.022 min-1 and ke = 0.043 +/- 0.012 min-1, respectively, suggesting that the process of bFGF internalization is not dramatically altered by HSPG. bFGF stimulated DNA synthesis to the same maximal level under both conditions, but chlorate-treated cells were significantly less responsive at low bFGF doses (approximately 10-fold increase in ED50). The differences observed for control and chlorate-treated cells in the dose-response curves for stimulation of DNA synthesis and receptor binding correlated directly, suggesting that receptors are equally capable of eliciting a mitogenic signal under both conditions. It is unlikely that these results are due to residual HSPG since heparinase (I and III) digestion of chlorate-treated cells had little effect. Although the presence of HSPG on the cell surface increases the affinity of bFGF for its receptors, our observations suggest that HSPG are not "absolutely" required for binding, internalization, or stimulation of mitogenic activity.
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PMID:Basic fibroblast growth factor binds its receptors, is internalized, and stimulates DNA synthesis in Balb/c3T3 cells in the absence of heparan sulfate. 866 12

Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.
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PMID:Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli. 878 Jun 85

We report evidence that gene complexes, consisting of polycations and plasmid DNA enter cells via binding to membrane-associated proteoglycans. Treatment of HeLa cells with sodium chlorate, a potent inhibitor of proteoglycan sulfation, reduced luciferase expression by 69%. Cellular treatment with heparinase and chondroitinase ABC inhibited expression by 78% and 20% with respect to control cells. Transfection was dramatically inhibited by heparin and heparan sulfate and to a smaller extent by chondroitan sulfate B. Transfection of mutant, proteoglycan deficient Chinese hamster ovary cells was 53 x lower than of wild-type cells. For each of these assays, the intracellular uptake of DNA at 37 degrees C and the binding of DNA to the cell membrane at 4 degrees C was impaired. Preliminary transfection experiments conducted in mutant and wild-type Chinese hamster ovary cells suggest that transfection by some cationic lipids is also proteoglycan dependent. The variable distribution of proteoglycans among tissues may explain why some cell types are more susceptible to transfection than others.
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PMID:Evidence for the role of proteoglycans in cation-mediated gene transfer. 890 84

Heparinases I, II and III from F. heparinum cleave heparin-like molecules, with a high degree of substrate specificity, at the glucosamine-uronate linkage by elimination, leaving an unsaturated C4-C5 bond in the uronic acid. The primary sequences of these enzymes have been reported earlier. In this study we perform a comparative analysis of the properties and primary sequences of heparinase I, II and III. Alignment of the primary sequences revealed little sequence homology (15% residue identity in a LALIGN alignment) at both DNA and amino acid levels. There are three basic clusters in heparinase II satisfying the heparin binding consensus sequence with one of the sequences sharing homology with a consensus sequence in the heparin binding site of heparinase I and two basic clusters in heparinase III. Similar to heparinase I, there are two putative 'EF-hand' calcium coordinating motifs in heparinase III, while heparinase II does not contain any such motifs. Recombinant heparinases II and III's degradation of the substrate and the subsequent separation of the oligosaccharide products by POROS anion exchange chromatography were identical to those obtained from native heparinases II and III from F. heparinum.
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PMID:A comparative analysis of the primary sequences and characteristics of heparinases I, II, and III from Flavobacterium heparinum. 895 71

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA or hepatitis C virus (HCV) RNA has been facilitated by adapting a spin column procedure for sample preparation and the use of chemiluminescent detection of polymerase chain reaction (PCR) products in microtiter plate format. All materials were commercially available and relatively inexpensive. By making a single dilution prior to amplification, concentrations of 500 copies to 2.5 million HIV-1 1 RNA copies per mL and 1,000 copies to 50 million HCV RNA copies per mL could be determined on 140-microL samples. Between-run imprecision employing the improved procedure for HIV-1 RNA was 23%. Correlation of HIV-1 RNA concentrations obtained using chemiluminescent detection with values obtained by colorimetric assay of PCR products was 0.98. Correlation of HCV RNA concentration determined by the spin column-chemiluminescent assay procedure with those obtained by branched DNA methodology was 0.91. Spin columns could be used with serum or plasma containing acid-citrate-dextrose or heparin anticoagulant, but heparinized samples required treatment with heparinase prior to amplification.
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PMID:Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products. 898 50

Heparin is a naturally occurring polysaccharide found primarily in the liver, lung and artery walls (White et al. 1968), which is commonly used as an anticoagulant for venal blood samples. Although the inhibitory effect of heparin on the polymerase chain reaction (PCR) and other enzyme-mediated reactions has been noted (Beutler et al. 1990; Izraeli et al. 1991), this is not widely known to field biologists collecting samples which are subsequently used for genetic analysis. The enzyme heparinase (Linker & Hovingh 1972) has been used successfully to eliminate heparin inhibition from DNA samples post-extraction, because the normal range of DNA extraction techniques fail to do so (Beutler et al. 1990; Oberhauser et al. 1994; Taylor et al. 1994). However, the cost of heparinase treatment for large numbers of samples, such as those from a population survey, would prove prohibitively expensive if the concentration and grade of heparinase (heparinase II) used by Beutler et al. (1990) is followed (see Table 1): heparinase II is more than three times the price per unit than heparinase I. If the blood-to-heparin ratio is relatively high during the original collection, it appears that the inhibitory effect of heparin can be diluted out. In addition DNA extracted from washed lymphocytes rather than whole blood may not show the same level of inhibition. For example, the DNA extracted from washed lymphocyte pellets from 10 mL heparinized mammalian blood samples is amplifiable in our laboratory. However, DNA extracts from small (less than 1 mL), frozen brushtail possum Trichosurus vulpecula blood samples collected into heparin fail to amplify in PCR reactions. The DNA was extracted by lysis of the whole blood (method in Taylor et al. 1991) in order to reduce the number of decanting steps in the procedure, because yields were expected to be low. I therefore carried out an experiment to examine the efficacy of different concentrations and grades of heparinase in the removal of presumed PCR inhibition from these samples.
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PMID:Titration of heparinase for removal of the PCR-inhibitory effect of heparin in DNA samples. 913 13

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


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