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)

The high-iron diamine staining (HID), which has been used in histochemistry to stain sulfated glycoconjugates (SGC), was tested for detectability of authentic acidic substances (chondroitin sulfates A plus C, dermatan sulfate, heparan sulfate, chondroitin, hyaluronic acid, alpha 1-acid glycoprotein and ribonucleic acid) in electrophoresis on cellulose acetate membrane (Separax). The results showed that only SGC were detectable by the HID, although all the above substances were stained with alcian blue. The glycoconjugate preparations obtained from the liver, kidney, lung, small intestine, colon, stomach, brain and spleen of rats were examined by two-dimensional electrophoresis on Separax. The new spots (or bands), besides those of sulfated glycosaminoglycans, were detected by the HID on the electrophoretograms of all the samples except for the kidney one. The substances giving the new spots (or bands) were indicated to be sulfated glycopeptides (SGP) by crude heparinase digestion of a representative sample. The present results revealed that the HID was applicable for detection of SGP in electrophoresis on cellulose acetate membrane. Also, it is a novel finding that the liver and spleen contain SGP.
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PMID:An application of the high-iron diamine staining for detection of sulfated glycoproteins (glycopeptides) in electrophoresis on cellulose acetate membrane. 240 56

We have isolated a syngeneic monoclonal antibody (HepSS-1) reactive to a murine methylcholanthrene-induced fibrosarcoma, Meth-A. HepSS-1 also bound to a wide variety of established and fresh normal cells derived from not only mice but also other species such as human, monkey, rat, hamster, and chicken. Immunoprecipitation of surface iodinated Meth-A cell extract with HepSS-1, as well as Sepharose 4B gel chromatography of Meth-A cell extract and detection of antigens recognized by HepSS-1 by a sandwich-type radioimmunoassay revealed that the HepSS-1 antigens were composed of several molecular species, with one as large as approximately 10(6) daltons. The following evidence indicates that HepSS-1 specifically recognizes an epitope present in heparan sulfate glycosaminoglycan (HS-GAG). First, treatment of Meth-A cells with heparitinase or heparinase, but not with chondroitinase ABC or hyaluronidase, resulted in the loss of HepSS-1 binding. Second, HS-GAG but not seven other types of GAG (hyaluronic acid, heparin, chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and keratan sulfate) inhibited HepSS-1 binding to Meth-A cells. Third, HepSS-1 bound with HS-GAG but not with the seven other types of GAG. From the binding analysis of HepSS-1 to various modified HS-GAG and whale omega-heparin, it is additionally suggested that HepSS-1 recognizes an epitope closely related to O-sulfated and N-acetylated glucosamine. We found that NIH 3T3 cells expressed more HepSS-1 epitopes at a low cell density than at confluency and in G2 + M than in G1, whereas NIH 3T3 cells transformed with Kirsten-ras oncogene or SV-40 expressed high levels of HepSS-1 epitopes and ceased to show the density-dependent change in the amount of HepSS-1 epitopes. These observations were also reproduced by using NIH 3T3 cells transformed with a temperature sensitive Kirsten murine sarcoma virus maintained at permissive and non-permissive temperatures. Thus HepSS-1 is a first monoclonal antibody to HS-GAG and seems to be useful to elucidate changes in cell surface HS-GAG in normal cell growth and cell transformation.
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PMID:A syngeneic monoclonal antibody to murine Meth-A sarcoma (HepSS-1) recognizes heparan sulfate glycosaminoglycan (HS-GAG): cell density and transformation dependent alteration in cell surface HS-GAG defined by HepSS-1. 243 Oct 47

The basement membranes of the innervated surface of the electric organ of Discopyge tschudii present a high concentration of mucopolysaccharides as revealed by intense ruthenium red-positive reaction. Glycosaminoglycans (GAGs) were isolated and characterized from these pure basement membranes by using a combination of agarose gel electrophoresis and enzymatic degradation with specific enzymes. The isolated basement membrane showed a high concentration of GAGs (130 mg/g of dry tissue); of this amount 49% was hyaluronic acid, 24% was chondroitin-6-sulfate, 12% was heparan sulfate, and 14% was dermatan sulfate. Controlled digestion with heparinase and heparitinases I and II was used to study the structural features of the heparan sulfate. Four unsaturated disaccharide units were found in the heparan sulfate: disulfated, N-sulfated, N-acetylated, and N-acetylated O-sulfated disaccharides. The disaccharide units of the cholinergic heparan sulfate present a high amount of disulfated disaccharides and a low amount of N-acetylated O-sulfated disaccharides. The N-sulfated disaccharides, in contrast to the N-acetylated ones, were found through all the structure of the cholinergic heparan sulfate. Finally our work shows for the first time the presence of dermatan sulfate in the basal lamina of the electric organ.
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PMID:Glycosaminoglycan composition of electric organ basement membranes. 243 1

To gain insight into the cellular and molecular mechanisms underlying cell interactions in the early postnatal mouse cerebellum, Ca2+-dependent and -independent aggregation mechanisms were characterized using single cell suspensions under conditions that allow discrimination between the two mechanisms. When cerebellar cells were derived from newborn to 10-day-old mouse cerebellum, both mechanisms were active and showed no major change in activity during this time period. Mg2+ could not replace Ca2+ in the Ca2+-dependent mechanism. In contrast to the Ca2+-independent mechanisms, the Ca2+-dependent mechanism was inactive at low temperatures, suggesting a necessity for molecular rearrangement within the surface membrane during aggregation. Neuraminidase, chondroitinase, heparinase or hyaluronidase treatment of cells did not influence the aggregation of cells under Ca2+-dependent and -independent conditions. Chondroitin sulfate inhibited and hyaluronic acid stimulated the Ca2+-dependent mechanism, whereas chondroitin sulfate only slightly and hyaluronic acid strongly inhibited the Ca2+-independent one. Dextran sulfate slightly inhibited both mechanisms, whereas heparin and fucoidan, a complex sulfated carbohydrate, did not influence cell aggregation, while they strongly inhibited attachment of cells to laminin. The polycation poly-L-lysine slightly stimulated the Ca2+-independent mechanism, but inhibited the Ca2+-dependent one. Interestingly, chondroitin sulfate and hyaluronic acid strongly stimulated cell aggregation under conditions where both mechanisms were almost destroyed or inactive. Dextran sulfate showed only a small effect under these conditions. These observations indicate that different molecular mechanisms are active in cell-cell versus cell-extracellular matrix interactions and suggest a hitherto unknown complexity in molecular mechanisms during early postnatal cerebellar development.
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PMID:Characterization of Ca2+-dependent and -independent aggregation mechanisms among mouse cerebellar cells. 246 13

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.
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PMID:The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers. 294 1

Methods for the analysis of urinary GAGs that can be used for or are applicable to routine assays are described. The most popular method for isolation of GAGs from a urine sample is CPC precipitation, in spite of the fact that it is time-consuming. To identify the different types of GAGs excreted, separation by one-dimensional cellulose acetate electrophoresis followed by staining with alcian blue or toluidine blue may suffice for routine purposes. Solvents such as barium acetate, calcium acetate, barbital buffer and pyridine-formic acid are used for the separation. However, the separation of the seven types of GAGs by conventional one-dimensional electrophoresis is difficult, and a discontinuous electrophoretic method with barium acetate buffer and barium acetate buffer containing ethanol has proved effective for the separation. HPLC separation methods are used for assaying the profiles of enzymatic digestion products of GAGs. Advanced HPLC methods for separating intact GAGs of different types are currently unavailable. Unsaturated disaccharides produced with heparitinase and/or heparinase from heparan sulphate and oligosaccharides produced by hyaluronidase digestion of hyaluronic acid can be separated by HPLC. For chondroitin sulphate isomers, unsaturated disaccharides produced by digestion of the samples with chondroitinase ABC or chondroitinase AC are separated by HPLC and determined by their UV absorbance or by fluorescence labelling. Highly sensitive quantitation of chondroitin sulphate isomers is possible by these methods, which are also efficient for the investigation of the constituents of GAG polymers. Some of these methods have been applied to urine samples from patients with, e.g., mucopolysaccharidoses.
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PMID:Methods for analysis of urinary glycosaminoglycans. 306 22

An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.
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PMID:Characterization of a cytosolic inhibitor of calf estrogen receptor binding to nuclei. 330 79

An in vitro model is presented for the study of glycosaminoglycans in human skin. The synthesis of six glycosaminoglycan species in both dermis and epidermis was measured by D-[3H]glucosamine labelling. Punched biopsies (epidermis + entire dermis) of 3 mm in diameter were cultured at 37 degrees C in 5% carbon dioxide-95% air. When the label was added 18 h after explantation, the incorporation started immediately, and for all glycosaminoglycans the time-dependent incorporation was linear for 16 h. The experimental variation was minimized by expressing the measurements in epidermis "per explant" and in dermis "per mg of wet explant". A ratio to dermal hydroxyproline did not improve the precision. Most of the variation arose "before" isolation and separation of the glycosaminoglycans. The labelled products were macromolecules and were converted to small molecules by chondroitinase ABC + heparinase. The total incorporation in dermis was 2 1/2 times higher than in epidermis. Hyaluronic acid was the predominant synthesized product in dermis, and hyaluronic acid and heparan sulphate were the predominant products in epidermis. The proportions (%) in dermis/epidermis were as follows: hyaluronic acid, 61/44; heparan sulphate, 18/31; dermatan sulphate, 5/8; chondroitin 4/6-sulphate, 10/7 and heparin-like glycosaminoglycan, 1/2. The same species were also demonstrated as native constituents of uncultured human skin. Hyaluronic acid and dermatan sulphate predominated in dermis, whereas no single species predominated in epidermis. Their concentrations in uronic acid equivalents per mg of wet skin (pmol/mg of epidermis + dermis) were as follows in dermis/epidermis: hyaluronic acid, 243/0.48; heparan sulphate, 22/0.44; dermatan sulphate, 170/0.56; chondroitin 4/6-sulphate, 72/0.50; and heparin-like glycosaminoglycan, 5/0.22. Thus, only 0.4% of the in vivo synthesized glycosaminoglycan was present in epidermis.
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PMID:D-[3H]glucosamine labelling of epidermal and dermal glycosaminoglycans in cultured human skin. 338 61

TA3 murine ascites adenocarcinoma cells were compared for their ability to release radioactive glucosamine and 35SO4-labeled glycoproteins and glycosaminoglycans into the culture medium. Both TA3-Ha and TA3-St cells contained cell-surface heparan sulfate that was released into culture, but not chondroitin sulfate. Both cells released a membranous aggregate of labeled components from the cell surface and hyaluronic acid from inside the cells that fractionated in the void volume of Sepharose CL-4B. This void-volume fraction from the TA3-Ha cells contained glucosamine-labeled epiglycanin at a higher concentration relative to other glucosamine-labeled components than that found on plasma membranes. Glycoproteins associated with epiglycanin found on the cell surface, as well as released into culture medium, contained sulfate that could not be removed by chondroitinase ABC, heparinase, or keratinase. Kinetic analysis of the glucosamine-labeled material released from TA3-Ha cells indicated that hyaluronic acid was released rapidly with a 45-min half-life, whereas the other membranous components were released much more slowly.
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PMID:Further characterization of the glycoproteins and glycosaminoglycans released from TA3 murine adenocarcinoma cells in culture. 376 85

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.
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PMID:Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney. 615 76


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