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 Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties

1989 ◽  
Vol 259 (1) ◽  
pp. 209-216 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Glucuronic Acid ◽  
Heparan Sulphate ◽  
Gel Permeation Chromatography ◽  
Electrophoretic Analysis ◽  
Native Molecular Mass ◽  
Phosphate Ions ◽  
Column Procedure ◽  
Inhibited Enzyme

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

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 Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

scholarly journals Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

2000 ◽  
Vol 345 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Bruno ANTONSSON ◽  
Sylvie MONTESSUIT ◽  
Sandra LAUPER ◽  
Robert ESKES ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Molecular Mass ◽  
Lipid Membranes ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Cytochrome C Release ◽  
Octyl Glucoside ◽  
Apoptotic Activity

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

scholarly journals Purification and characterization of casein kinase I from broccoli

1993 ◽  
Vol 293 (1) ◽  
pp. 283-288 ◽  
Author(s):  
L J Klimczak ◽  
A R Cashmore
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Native Molecular Mass ◽  
Specificity And Sensitivity ◽  
Sds Page

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

scholarly journals Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization

1991 ◽  
Vol 279 (2) ◽  
pp. 515-520 ◽  
Author(s):  
J Bielicki ◽  
J J Hopwood
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Chloride Ions ◽  
Lysosomal Degradation ◽  
Purification And Characterization ◽  
Keratan Sulphate ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Molecular Masses ◽  
Column Procedure

Human N-acetylgalactosamine 6-sulphatase (EC 3.1.6.14), which is involved in the lysosomal degradation of the glycosaminoglycans keratan sulphate and chondroitin 6-sulphate, was purified more than 130,000-fold in 2.8% yield from liver by an eight-step column procedure. One major form was identified with a pI of 5.7 and a native molecular mass of 62 kDa by gel filtration. When analysed by SDS/PAGE, dithioerythritol-reduced enzyme contained polypeptides of molecular masses 57 kDa, 39 kDa and 19 kDa, whereas non-reduced enzyme contained a major polypeptide of molecular mass 70 kDa. It is proposed that active enzyme contains either the 57 kDa polypeptide or disulphide-linked 39 kDa and 19 kDa polypeptides. Minor amounts of other enzyme forms separated during the chromatofocusing step and the Blue A-agarose step were not further characterized. Purified N-acetylgalactosamine 6-sulphatase was inactive towards 4-methylumbelliferyl sulphate, but was active, with pH optima of 3.5-4.0, towards 6-sulphated oligosaccharide substrates. Km values of 12.5 and 50 microM and Vmax. values of 1.5 and 0.09 mumol/min per mg were determined with oligosaccharide substrates derived from chondroitin 6-sulphate and keratan sulphate respectively. Sulphate, phosphate and chloride ions were inhibitors of enzyme activity towards both substrates, with 50 microM-Na2SO4 giving 50% inhibition towards the chondroitin 6-sulphate trisaccharide substrate.

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 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 Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

1997 ◽  
Vol 326 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Sergio LIZANO ◽  
Bruno LOMONTE ◽  
Jay W. FOX ◽  
José Maréa GUTIÉRREZ
Keyword(s):  
Phospholipase A2 ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Biological Activities ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Cytolytic Activity ◽  
Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

scholarly journals Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain

1990 ◽  
Vol 270 (3) ◽  
pp. 715-719 ◽  
Author(s):  
A Höer ◽  
D Höer ◽  
E Oberdisse
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Kinetic Data ◽  
Gel Filtration ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Gel Filtration Chromatography ◽  
Pyrimidine Nucleotides ◽  
High Affinity ◽  
Marked Inhibition

We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.

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