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)

When cultures of endothelial cells prelabeled with H2 -35-SO4 are exposed to a purified preparation from induced Flavobacterium heparinum containing heparinase and heparitinase activities, radioactivity accumulates in the supernatant medium. After further treatment in vitro with crude enzyme this material migrates, in part, as glucosamine (N,O-disulfated glucosamine), a break-down product characteristic of heparin and heparin-related mucopolysaccharides. After exposure of the cultures to the purified enzyme, the amount of acid-insoluble -3 5-S radioactivity that can be removed with EDTA is decreased compared to that that can be removed from control cultures. Since the amount of radioactivity that is released as break-down products is much higher than the amount of radioactivity that is secreted into the supernatant medium as intact (non-dialysable) mucopolysaccharide chains in control plates, the action of the enzyme appears to be on the cell itself. The data presented support previous studies suggesting that chains of heparitin sulfate that are accessible to the action of the enzyme are present at the surface of endothelial cells.
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PMID:Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures. 12 75

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.
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PMID:Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b. 50 Dec 88

The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95). 160 62

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.
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PMID:Inhibition of human immunodeficiency virus gene amplification by heparin. 190 9

Preincubation of Vero cells with 1 microM phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-(DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl)-L-3-(2' - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.
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PMID:Diphtheria toxin endocytosis and membrane translocation are dependent on the intact membrane-anchored receptor (HB-EGF precursor): studies on the cell-associated receptor cleaved by a metalloprotease in phorbol-ester-treated cells. 764 57

Amplification by the polymerase chain reaction (PCR) of hepatitis B virus (HBV) DNA extracted from parallel samples of serum and heparinized plasma gave contradictory results, indicating that heparin inhibits virus detection. Similarly, analysis of PCR products of woodchuck hepatitis virus (WHV) DNA showed that heparinization of blood abolished WHV DNA amplification, while anticoagulation with sodium EDTA or acid citrate dextrose did not. Amplification of recombinant WHV and HBV DNA in the presence of increasing concentrations of sodium heparin progressively inhibited and finally abolished virus genome detection. The inhibitory effect of heparin was reversed by treatment of either plasma or isolated DNA with heparinase (5 U/reaction, 1 h at 28 degrees C) prior to PCR. In contrast, heparin did not influence the detection of hepadnavirus in peripheral blood mononuclear cells (PBMC), even after prolonged incubation of the cells with heparin in culture. These findings confirm that heparin exerts a dramatic inhibitory effect on hepadnaviral DNA detection by PCR and they demonstrate that this effect can be reversed by heparinase. The findings also show that extensively washed PBMC derived from heparinized blood can be a reliable source of nucleic acids for amplification of hepadnavirus genome. These results imply that previous data should be reassessed if samples of heparinized plasma were found hepadnavirus DNA nonreactive by PCR or when these samples were used as a starting material for PCR quantitation of viral genome.
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PMID:Detection of hepatitis B and woodchuck hepatitis viral DNA in plasma and mononuclear cells from heparinized blood by the polymerase chain reaction. 773 48

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

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.
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PMID:Characterization of a particulate pathway for copper in K562 cells. 813 Feb 71

Binding of urinary protein C inhibitor (PCI) to cultured human epithelial kidney tumor cells (TCL-598) was studied. Binding was dose-dependent, time-dependent, and saturable. Heparin interfered in a dose-dependent way with PCI binding to TCL-598 as did heparan sulfate and to a lesser degree also dermatan sulfate. Pretreatment of TCL-598 with protamine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and > 100 micrograms/ml protamine sulfate reduced binding of PCI to < 10% of the control. Binding of 125I-PCI was specific, and bound 125I-PCI was recovered from the cells by heparin treatment or detached together with intact cells by EDTA treatment, migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound 125I-PCI. Furthermore, cell-bound PCI was functionally active as judged from its ability to inhibit the amidolytic activity of urokinase, and its inhibitory activity was stimulated approximately 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the luminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of heparin (50 micrograms/ml) and after pretreatment of the cells either with protamine sulfate (400 micrograms/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cells with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approximately 70% after chondroitinase ABC treatment of the extracellular matrices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximately 95%. These data suggest that heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfate-containing proteoglycans, the overall predominant PCI-binding proteoglycans in TCL extracts, are responsible for PCI binding to the extracellular matrix. Heparan sulfate, however, exposed to an environment containing PCI under physiological conditions, might localize PCI and modulate its target enzyme specificity in vivo.
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PMID:Binding of urinary protein C inhibitor to cultured human epithelial kidney tumor cells (TCL-598). The role of glycosaminoglycans present on the luminal cell surface. 818 78

The alphaherpesvirus pseudorabies virus (PrV) has been shown to attach to cells by interaction between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). A secondary binding step requires gD and presumably another, hitherto unidentified cellular receptor. By use of a virus overlay protein binding assay (VOPBA), cosedimentation analyses, and affinity chromatography, we identified three species of cell membrane constituents that bind PrV. By treatment with EDTA, peripheral HSPGs of very high apparent molecular mass (>200 kDa) could be extracted from Madin-Darby bovine kidney cells. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzymatic digestion with heparinase or papain. Cosedimentation analyses indicated that binding between PrV and high-molecular-weight HSPG depended on the presence of gC in the virion. In addition, adsorption of radiolabeled PrV virions to cells could be inhibited by the addition of purified high-molecular-weight HSPG. By using urea extraction buffer, a second species of HSPG of approximately 140 kDa could be solubilized. Binding of PrV to this HSPG in the VOPBA was also dependent on the presence of heparan sulfate, since reactivity was abolished after suppression of glycosaminoglycan biosynthesis with NaClO3 and after heparinase treatment. In addition to HSPG, in cellular membrane extracts obtained by treatment with mild detergent, a 85-kDa membrane protein was demonstrated to bind PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents that bind PrV: a peripheral HSPG of high molecular weight, an integral HSPG of approximately 140 kDa, and an integral membrane protein of 85 kDa. It is tempting to speculate that interaction between PrV and the two species of HSPG mediates primary attachment of PrV and that the 85-kDa protein is involved in a subsequent attachment step.
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PMID:Identification of cell surface molecules that interact with pseudorabies virus. 864 35


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