scholarly journals Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure

1991 ◽  
Vol 273 (3) ◽  
pp. 553-559 ◽  
Author(s):  
J E Turnbull ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Average Length ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Central Position ◽  
Human Skin Fibroblast ◽  
Cleavage Sites ◽  
Average Molecular Mass ◽  
Polymeric Structure

The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

scholarly journals Human platelet heparanase: purification, characterization and catalytic activity

1998 ◽  
Vol 330 (3) ◽  
pp. 1341-1350 ◽  
Author(s):  
Craig FREEMAN ◽  
R. Christopher PARISH
Keyword(s):  
Molecular Mass ◽  
Tumour Cell ◽  
Human Platelet ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Aggregation ◽  
Capillary Endothelium ◽  
Gel Filtration Chromatography ◽  
Average Molecular Mass ◽  
Tumour Metastasis

Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C. R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.

scholarly journals Nitric oxide degradation of heparin and heparan sulphate

1997 ◽  
Vol 324 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Rolando E. VILAR ◽  
Dineshchandra GHAEL ◽  
Min LI ◽  
Devan D. BHAGAT ◽  
Lisa M. ARRIGO ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mass ◽  
Inflammatory Responses ◽  
Degradation Products ◽  
Bone Development ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
No Synthase ◽  
Strong Anion Exchange

NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

scholarly journals Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane

1989 ◽  
Vol 264 (2) ◽  
pp. 457-465 ◽  
Author(s):  
L P W J van den Heuvel ◽  
J van den Born ◽  
T J A M van de Velden ◽  
J H Veerkamp ◽  
L A H Monnens ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Molecular Mass ◽  
Core Protein ◽  
Mammalian Species ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Heparan Sulphate Proteoglycan ◽  
Average Molecular Mass ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.

scholarly journals Purification, Characterization and Degradation Performance of a Novel Dextranase from Penicillium cyclopium CICC-4022

2019 ◽  
Vol 20 (6) ◽  
pp. 1360 ◽  
Author(s):  
Ruijie Huang ◽  
Lei Zhong ◽  
Fengwei Xie ◽  
Liming Wei ◽  
Lanfang Gan ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Metal Ion ◽  
Specific Activity ◽  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Fractional Precipitation ◽  
Penicillium Species ◽  
Average Molecular Mass ◽  
Penicillium Cyclopium ◽  
Effects Of Temperature

A novel dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractional precipitation and gel filtration chromatography. The effects of temperature, pH and some metal ions and chemicals on dextranase activity were investigated. Subsequently, the dextranase was used to produce dextran with specific molecular mass. Weight-average molecular mass (Mw) and the ratio of weight-average molecular mass/number-average molecular mass, or polydispersity index (Mw/Mn), of dextran were measured by multiple-angle laser light scattering (MALS) combined with gel permeation chromatography (GPC). The dextranase was purified to 16.09-fold concentration; the recovery rate was 29.17%; and the specific activity reached 350.29 U/mg. Mw of the dextranase was 66 kDa, which is similar to dextranase obtained from other Penicillium species reported previously. The highest activity was observed at 55 °C and a pH of 5.0. This dextranase was identified as an endodextranase, which specifically degraded the α-1,6 glucosidic bonds of dextran. According to metal ion dependency tests, Li+, Na+ and Fe2+ were observed to effectively improve the enzymatic activity. In particular, Li+ could improve the activity to 116.28%. Furthermore, the dextranase was efficient at degrading dextran and the degradation rate can be well controlled by the dextranase activity, substrate concentration and reaction time. Thus, our results demonstrate the high potential of this dextranase from Penicillium cyclopium CICC-4022 as an efficient enzyme to produce specific clinical dextrans.

scholarly journals Determination of Cellulose Degree of Polymerization in Historical Papers with High Lignin Content

Polymers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 1990
Author(s):  
Jasna Malešič ◽  
Ida Kraševec ◽  
Irena Kralj Cigić
Keyword(s):  
Acetic Acid ◽  
Molecular Mass ◽  
Lignin Content ◽  
Degree Of Polymerization ◽  
Average Degree ◽  
Size Exclusion ◽  
Sodium Chlorite ◽  
Average Molecular Mass ◽  
High Lignin Content

Determination of cellulose degree of polymerization (DP) is one of the most commonly used methods in paper degradation studies, performed either by a standardized method using viscometry (as average degree of polymerization (DPv)) or size-exclusion chromatography (SEC) (as weight average molecular mass (Mw)). Due to the insolubility of papers with high lignin content in cupriethylenediamine (CED), such as groundwood papers, viscometric determination is not possible; therefore, pretreatment is required to allow subsequent dissolution of the papers. In this study, the pretreatment of historical papers containing groundwood with sodium chlorite in acetic acid was investigated, which enables dissolution of the paper samples in CED and determination of the cellulose average degree of polymerization by viscometry (DPv). Kappa number was determined to estimate the lignin content in the papers. The suitability of SEC UV-VIS analysis for determination of Mw in papers with high lignin content had been verified before it was used as a comparative method for viscometry. Using SEC, changes in the weight average molecular mass (Mw) of cellulose tricarbanilate (CTC) derivative during delignification were evaluated. The results indicate that no significant depolymerization occurred in the selected samples under the studied delignification conditions, which was additionally confirmed with determination of monosaccharides by ion chromatography. The results of the Mw determinations by SEC and DPv by viscometry are in good correlation, justifying the use of viscometry after chlorite/acetic acid pretreatment to determine the cellulose average degree of polymerization in historical papers with high lignin content.

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 Investigations into the molecular size and shape of tomato extensin

1996 ◽  
Vol 320 (2) ◽  
pp. 577-583 ◽  
Author(s):  
Michael D. BROWNLEADER ◽  
Olwyn BYRON ◽  
Arthur ROWE ◽  
Michael TREVAN ◽  
Kevin WELHAM ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Plant Disease Resistance ◽  
Average Length ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Primary Cell ◽  
Sedimentation Equilibrium ◽  
Primary Cell Wall ◽  
Molecular Characteristics

The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457–469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40–50 nm in length and 2.0–2.5 nm in width. A minority of these particles showed a central ‘kink’. A number of smaller and generally wider particles (20 nm×2–4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer–dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 µM and sedimentation velocity data generated a Kd between 1 and 10 µM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.

Extraction of polyethylene glycols with dicarbolide solutions in nitrobenzene

1990 ◽  
Vol 55 (8) ◽  
pp. 1959-1967 ◽  
Author(s):  
Petr Vaňura ◽  
Pavel Selucký
Keyword(s):  
Polyethylene Glycol ◽  
Molecular Mass ◽  
Extraction Constant ◽  
Metal Extraction ◽  
Polyethylene Glycols ◽  
Relative Molecular Mass ◽  
Published Data ◽  
Average Molecular Mass ◽  
Peg 400 ◽  
Extraction Constants

The extraction of polyethylene glycol of average molecular mass 400 (PEG 400) with dicarbolide solution in nitrobenzene and of longer-chain polyethylene glycol, of average molecular mass 1 500 (PEG 1 500), with chlorinated dicarbolide solution in nitrobenzene was studied. During the extraction of PEG 400, the polyethylene glycol solvates the Horg+ ion in the organic phase giving rise to the HLorg+ species (L is polyethylene glycol). The obtained value of the extraction constant Kex(HLorg+) = 933 is consistent with published data of metal extraction. Extraction of PEG 1 500 was treated applying the simplified assumption that the thermodynamic behaviour of PEG 1 500 is the same as that of n molecules of polyethylene glycol with relative molecular mass 1 500/n, each solvating one cation. For this model, the value of n = 3.2 ± 1.1 and the values of the extraction constants of the HL1/n,org+ and HL2/n,org+ species were obtained by using the adapted program LETAGROP. This value of n is consistent with published extraction data in the presence of polyethylene glycol with a relative molecular mass from 200 to 1 000.

scholarly journals Nanomechanical Molecular Mass Sensing Using Suspended Microchannel Resonators

Sensors ◽  
2021 ◽  
Vol 21 (10) ◽  
pp. 3337
Author(s):  
Alberto Martín-Pérez ◽  
Daniel Ramos ◽  
Javier Tamayo ◽  
Montserrat Calleja
Keyword(s):  
Molecular Mass ◽  
Mass Density ◽  
Applied Pressure ◽  
Atomic Mass ◽  
Liquid Sample ◽  
Mass Sensing ◽  
Average Molecular Mass ◽  
Fluid Properties ◽  
Molecular Masses ◽  
Microchannel Resonators

In this work we study the different phenomena taking place when a hydrostatic pressure is applied in the inner fluid of a suspended microchannel resonator. Additionally to pressure-induced stiffness terms, we have theoretically predicted and experimentally demonstrated that the pressure also induces mass effects which depend on both the applied pressure and the fluid properties. We have used these phenomena to characterize the frequency response of the device as a function of the fluid compressibility and molecular masses of different fluids ranging from liquids to gases. The proposed device in this work can measure the mass density of an unknown liquid sample with a resolution of 0.7 µg/mL and perform gas mixtures characterization by measuring its average molecular mass with a resolution of 0.01 atomic mass units.

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