scholarly journals Inhibition by heparin-modulated antithrombin III of amidolysis catalysed by mβ-acrosin

1984 ◽  
Vol 217 (3) ◽  
pp. 721-725
Author(s):  
W F Long ◽  
F B Williamson
Keyword(s):  
Hyaluronic Acid ◽  
Molecular Mass ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Relative Molecular Mass ◽  
Dextran Sulphate ◽  
Dermatan Sulphate ◽  
Arginine Residues ◽  
Cellulose Sulphate

Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.

scholarly journals Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties

1990 ◽  
Vol 271 (1) ◽  
pp. 75-86 ◽  
Author(s):  
J Bielicki ◽  
C Freeman ◽  
P R Clements ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Human Kidney ◽  
Reduced Form ◽  
Dermatan Sulphate ◽  
Native Molecular Mass ◽  
Phosphate Ions

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

scholarly journals Pregnancy-related changes in rat cervical glycosaminoglycans

1980 ◽  
Vol 192 (1) ◽  
pp. 1-8 ◽  
Author(s):  
A M Golichowski ◽  
S R King ◽  
K Mascaro
Keyword(s):  
Hyaluronic Acid ◽  
Gestational Age ◽  
Uronic Acid ◽  
Heparan Sulphate ◽  
Late Pregnancy ◽  
Dermatan Sulphate ◽  
Collagen Fibres ◽  
Cellulose Acetate Electrophoresis ◽  
Pregnant Rats ◽  
Pregnant State

Non-pregnant and pregnant rats of known gestational age were killed at intervals and their uterine cervices were excised and digested with papain. Glycosaminoglycans thus extracted were separated by cellulose acetate electrophoresis and stained with Alcian Blue. Glycosaminoglycans were identified by comparison with standards and by serial degradation with chondroitin ABC lyase, butyl nitrite and leech hyaluronidase. Dermatan sulphate, hyaluronic acid and heparan sulphate were identified and quantitative determined by densitometry. The overall concentration of glycosaminoglycans changed little during pregnancy. A 3-fold total increase in uronic acid paralleled the increase in cervical weight. Hyaluronate content, however, increased 17-fold, and rose from 6% of total glycosaminoglycans in the non-pregnant state to 33% at term. Furthermore, the ratio of hyaluronate to hydroxyproline increased 10-fold. These changes are consistent with an accumulation of hyaluronate in the interstices between collagen fibres, resulting in the softening of this tissue that is seen in late pregnancy.

scholarly journals Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

1980 ◽  
Vol 190 (2) ◽  
pp. 243-254 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Hyaluronic Acid ◽  
Salt Concentration ◽  
Heparan Sulphate ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan ◽  
Spent Media

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.

scholarly journals Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells

1978 ◽  
Vol 172 (3) ◽  
pp. 443-456 ◽  
Author(s):  
B Glimelius ◽  
B Norling ◽  
B Westermark ◽  
Å Wasteson
Keyword(s):  
Hyaluronic Acid ◽  
Malignant Glioma ◽  
Membrane Fraction ◽  
Heparan Sulphate ◽  
Glioma Cells ◽  
Gel Chromatography ◽  
Total Production ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Malignant Glioma Cells

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.

The effect of 1-thyroxine treatment on skin accumulation of acid glycosaminoglycans in primary myxoedema

1986 ◽  
Vol 113 (1) ◽  
pp. 56-58 ◽  
Author(s):  
P. Lund ◽  
K. Hørslev-Petersen ◽  
P. Helin ◽  
H.-H. Parving
Keyword(s):  
Hyaluronic Acid ◽  
Binding Capacity ◽  
Heparan Sulphate ◽  
Dermatan Sulphate ◽  
Water Binding ◽  
Control Value ◽  
Thyroxine Treatment ◽  
Skin Accumulation ◽  
Skin Biopsies ◽  
Acid Glycosaminoglycans

Abstract. We assessed skin accumulation of acid glycosaminoglycans in ten patients with primary myxoedema (median age 70 years) before and during 1-thyroxine treatment (median 7 months). Eight subjects matched for sex and age served as controls. Acid glycosaminoglycans were determined biochemically on small skin biopsies. Hyaluronic acid was elevated in untreated myxoedema, median 0.60, range 0.39–0.90 μg hexosamine/mg dried defatted tissue compared to a median control value of 0.52, range 0.43–0.65 μg hexosamine/mg dried defatted tissue, P < 0.02. Chondroitin-4,6-sulphate and dermatan sulphate showed no elevation, while heparan sulphate was actually reduced in myxoedema, P < 0.01. 1-thyroxine treatment induced a significant reduction in hyaluronic acid, median 0.41, range 0.24–0.71 μg hexosamine/mg dried defatted tissue, while no consistent changes occurred in the remaining three acid glycosamine glycans. The accumulation of hyaluronic acid might contribute to the peculiar non-pitting oedema of the skin in myxoedema, due to the strong water-binding capacity of the substance.

scholarly journals The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells

1976 ◽  
Vol 158 (3) ◽  
pp. 567-573 ◽  
Author(s):  
B Y J T Yue ◽  
J L Baum ◽  
J E Silbert
Keyword(s):  
Hyaluronic Acid ◽  
Stromal Cells ◽  
Heparan Sulphate ◽  
Deae Cellulose ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Monolayer Cultures ◽  
Sulphated Glycosaminoglycans ◽  
Cellulose Column ◽  
Sulphated Glycosaminoglycan

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.

Potentiation of Antithrombin III Activity by Sulphated Polysaccharides

1979 ◽  
Author(s):  
G. Kindness ◽  
W.F. Long ◽  
F.B. Williamson
Keyword(s):  
Antithrombin Iii ◽  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Relative Effectiveness ◽  
Coagulation Factor ◽  
Factor X ◽  
Sulphated Polysaccharides ◽  
Molecular Rigidity ◽  
Cellulose Sulphate

Because of current interest in the mechanisms of antithrombin III-mediated anticoagulation by heparin, we examined the anticoagulant effectiveness of a number of other sulphated polysaccharides of relatively well-defined chemical structure using ex vivo assay systems. Our results suggest that heparan sulphate, cellulose sulphate, dextran sulphate, xylan sulphate, and a number of sulphated galactans derived from marine algae are able, like heparin, to potentiate antithrombin III inhibition of thrombin and activated coagulation factor X. Dermatan sulphate exerts anticoagulant activity which appears to be independent of antithrombin III. The relative effectiveness of these polymers as activators of antithrombin III may be related to their molecular rigidity.

scholarly journals Mechanism of inhibition of mammalian DNA topoisomerase I by heparin

1987 ◽  
Vol 241 (1) ◽  
pp. 111-119 ◽  
Author(s):  
K Ishii ◽  
S Futaki ◽  
H Uchiyama ◽  
K Nagasawa ◽  
T Andoh
Keyword(s):  
Topoisomerase I ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Structural Features ◽  
Dna Topoisomerase I ◽  
Dermatan Sulphate ◽  
Dna Topoisomerase ◽  
High Affinity ◽  
Mechanism Of Inhibition ◽  
Series Of Experiments

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.

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