scholarly journals Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties

1986 ◽  
Vol 234 (1) ◽  
pp. 83-92 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Human Liver ◽  
Incubation Temperature ◽  
Heparan Sulphate ◽  
Gel Permeation Chromatography ◽  
Substrate Affinity ◽  
Native Protein ◽  
Monosaccharide Residue ◽  
Ph Response

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.

scholarly journals Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties

1987 ◽  
Vol 246 (2) ◽  
pp. 355-365 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Carboxy Group ◽  
Human Liver ◽  
Catalytic Mechanism ◽  
Heparan Sulphate ◽  
Catalytic Efficiency ◽  
Keratan Sulphate ◽  
Ph Response ◽  
A Minor

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.

scholarly journals Human α-l-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate

1992 ◽  
Vol 282 (3) ◽  
pp. 899-908 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Human Liver ◽  
Skin Fibroblast ◽  
Catalytic Properties ◽  
Heparan Sulphate ◽  
Structural Features ◽  
Lysosomal Degradation ◽  
Catalytic Activities ◽  
Wide Range

The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.

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.

Proton MR Spectroscopic Features of the Human Liver: In-Vivo Application to the Normal Condition

1999 ◽  
Vol 40 (1) ◽  
pp. 77
Author(s):  
Soon Gu Cho ◽  
Mi Young Kim ◽  
Young Soo Kim ◽  
Won Choi ◽  
Seok Hwan Shin ◽  
...  
Keyword(s):  
Normal Condition ◽  
Human Liver

Developmental stages, incubation temperature, and in vivo traceability of primordial germ cell in an important aquaculture species Piaractus mesopotamicus

Aquaculture ◽  
2021 ◽  
Vol 535 ◽  
pp. 736381
Author(s):  
Geovanna Carla Zacheo Coelho ◽  
Dilberto Ribeiro Arashiro ◽  
Tamiris Disselli ◽  
Matheus Pereira-Santos ◽  
Tatiana María Mira-López ◽  
...  
Keyword(s):  
Germ Cell ◽  
Developmental Stages ◽  
Incubation Temperature ◽  
Primordial Germ Cell ◽  
Piaractus Mesopotamicus

Controlled Nano–Bio Interface of Functional Nanoprobes for in Vivo Monitoring Enzyme Activity in Tumors

ACS Nano ◽  
2019 ◽  
Author(s):  
Ziyan Sun ◽  
Kai Cheng ◽  
Yuyu Yao ◽  
Fengyu Wu ◽  
Jonathan Fung ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
In Vivo Monitoring

Nutrient uptake and enzyme activity of the preattachment goat embryo developed in vivo

1994 ◽  
Vol 41 (1) ◽  
pp. 204 ◽  
Author(s):  
D.K. Gardner ◽  
M. Lane ◽  
P.A. Batt
Keyword(s):  
Enzyme Activity ◽  
Nutrient Uptake

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

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