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)

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.
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PMID:Structural studies of heparitin sulfates. 12 78

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. The findings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific for heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent major components of these sites since the latter are not affected by digestion with neuraminidase and other glycosaminoglycan-specific enzymes. Identical findings were obtained on basement membranes in other locations (Bowman's capsule, tubule epithelium, and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.
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PMID:Presence of heparan sulfate in the glomerular basement membrane. 15 19

Glycosaminoglycans were isolated from purified fractions of glomerular basement membranes and partially characterized by chemical analysis and cellulose acetate electrophoresis. Basement membranes were prepared by detergent treatment of rat glomeruli and subjected to digestion with papain and Pronase. Glycosaminoglycans were isolated from the digests by precipitation with cetyl pyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated glycosaminoglycan fraction revealed the presence of one major and one minor spot. The major spot was identified as heparan sulfate because it comigrated with the heparan sulfate standard and was sensitive to heparinase and to nitrous acid oxidation but insensitive to chondroitinase ABC and to testicular or leech hyaluronidase. The minor spot was tentatively identified as hyaluronic acid based on its migratory behavior and sensitivity to leech and testicular hyaluronidase. The chemical composition of the isolated glycosaminoglycan was typical of that of heparan sulfate (high carbazole/orcinol ratio, high sulfate content, absence of galactosamine). The data support and confirm the cytochemical data obtained previously [Kanwar, Y. S. & Farquhar, M. G. (1979) Proc. Natl. Acad. Sci. USA 76, 1303-1307] demonstrating that heparan sulfate is the only sulfated glycosaminoglycan detectable in the glomerular basement membrane. The present results suggest that in addition to sulfated glycosaminoglycan some nonsulfated glycosaminoglycan (hyaluronic acid) may also be present in the glomerular basement membrane.
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PMID:Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. 15 57

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.
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PMID:Structure of the antithrombin-binding site in heparin. 22 60

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.
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PMID:Glycosaminoglycans of normal and malignant cultured human mammary cells. 42 76

Micro-scale isolation of sulfated glycopeptide from tissue was achieved by successive application of pronase digestion (Step 1), cetylpyridinium chloride-fractionation (Step 2), crude heparinase digestion or chondroitinase ABC digestion plus nitrous acid treatment (Step 3) and preparative cellulose acetate membrane-electrophoresis (Step 4). By this method, sulfated glycopeptide was obtained in a high yield from estrogen-treated rabbit uterus.
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PMID:A method for micro-scale isolation of sulfated glycopeptide from tissue. 52 51

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

The proteoglycans (PGs) in the guinea pig seminal vesicle were demonstrated ultrastructurally by both cuprolinic blue (CB) and ruthenium red (RR) staining. The PGs appeared as electron-dense granules with RR, but were filamentous following CB staining using the critical electrolyte concentration method. Three major types of PGs (T1, T2, T3) have been described according to their different locations and sizes. T1 filaments were short and were found mostly on both sides of the lamina densa of the basal lamina of the glandular epithelium (40-60 nm long) and also on the basal laminae of smooth muscle cells and capillary endothelial cells (20-30 nm long). In the epithelial basal lamina they were regularly spaced at an interval of 40-60 nm. T1 filaments in the lamina densa were smaller and more randomly distributed. Cytochemical characterisation of these PGs by various GAG degrading enzymes showed that T1 PGs are rich in heparan sulphate. T2 filaments were 30-40 nm long and closely associated with the collagen fibrils. They were arranged perpendicular to the long axis of collagen fibrils, also at intervals of about 60 nm. T2 filaments were removed by chondroitinase (Ch)-ABC, Ch-ABC plus Streptomyces (S)-hyaluronidase and pronase, but resistant to nitrous acid, heparitinase, heparinase, neuraminidase and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulphate. T3 filaments (60-100 nm) were widely distributed in the stroma at sites such as the interstitial spaces of the lamina propria, the reticular layer below the basal lamina, around individual collagen fibrils or bundles of such fibres, and on the cell surfaces of fibroblasts. The T3 filaments were removed by Ch-ABC, Ch-AC and pronase but were resistant to heparitinase, heparinase, S-hyaluronidase, neuraminidase and nitrous acid. They are therefore rich in chondroitin sulphate.
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PMID:Cytochemical localisation and characterisation of proteoglycans (glycosaminoglycans) in the epithelial-stromal interface of the seminal vesicle of the guinea pig. 128 Jun 36

In this paper we show that an anticoagulant activity, which we measure by thrombin time, appears in human plasma after its exhaustive proteolytic digestion. This activity is extremely heat stable, it is resistant to chondroitin ABC lyase (E.C.4.2.2.4) and heparan sulfate lyase (E.C.4.2.2.8), it is sensitive to heparin lyase (E.C.4.2.2.7) and to nitrous acid treatment: we suggest that it can be identified as authentic heparin. The amount present in 1 ml of plasma of healthy subjects corresponds to 0.1-0.2 I.U. of standard heparin (150 I.U./mg). Proteolytically digested human plasma was submitted to ion-exchange chromatography on DEAE-Sephacel and the anticoagulant activity in the fractions eluted at the different molarities of NaCl was measured by thrombin time. This analysis shows that the anticoagulant activity elutes at very low ionic strength. The possibility that interactions of the endogenous heparin with proteins or protein fragments are responsible for the difficulty in isolating heparin from human plasma is discussed.
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PMID:Endogenous heparinase-sensitive anticoagulant activity in human plasma. 144 May 19

The effect of specific glycosaminoglycan-hydrolyzing enzymes on the ruthenium red staining of pig spermatozoa was studied. Washed spermatozoa were incubated at 35 degrees C in buffer or with neuraminidase 0.5 units/ml, heparinase 0.2 mg/ml, or chondroitinase ABC 2.0 units/ml. After incubation sperm cells were washed, stained with ruthenium red and studied under the electron microscope. Anionic sites in the surface of untreated spermatozoa follow regularly the plasma membrane, but present are numerous processes constituting what has been defined as the glycocalyx. Neuraminidase did not affect the distribution of ruthenium red on the surface of the spermatozoa, but eliminated almost completely the processes of the glycocalyx. Heparinase caused loss of the ruthenium red-stained sites on the membrane surface of pig spermatozoa with less influence on the dense processes of the glycocalyx. A similar loss of ruthenium red-stained sites was observed with nitrous acid treatment. A striking effect of treatment with chondroitinase ABC was the production of a typical acrosome reaction.
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PMID:Glycosaminoglycan-sulfate as plasma membrane component of pig spermatozoa. 169 1


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