scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

scholarly journals Purification and properties of the hexosaminidase A-activating protein from human liver

1977 ◽  
Vol 167 (3) ◽  
pp. 693-701 ◽  
Author(s):  
Peter Hechtman ◽  
Dorothy LeBlanc
Keyword(s):  
Human Liver ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Hydrolase Activity ◽  
Gm2 Ganglioside ◽  
Purification And Properties ◽  
Hexosaminidase A ◽  
Hydrolysis Of

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of GM2 ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of GM2 ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the GM2 ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for GM2 ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-GM2 ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of GM2 ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme–activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1973 ◽  
Vol 131 (2) ◽  
pp. 381-388 ◽  
Author(s):  
F. Reyes ◽  
R. J. W. Byrde
Keyword(s):  
Nitrogen Source ◽  
Cellulose Acetate ◽  
Isoelectric Focusing ◽  
Culture Filtrate ◽  
Gel Filtration ◽  
Partial Purification ◽  
Conflicting Evidence ◽  
Purification And Properties ◽  
Km Value ◽  
Hydrolysis Of

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a β-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN′-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The Km value for hydrolysis of p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside at 37°C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.

scholarly journals Purification and properties of a kininogenin from the venom of Vipera ammodytes ammodytes

1976 ◽  
Vol 153 (2) ◽  
pp. 409-414 ◽  
Author(s):  
G S Bailey ◽  
R A Shipolini
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Vasoactive Peptides ◽  
Bovine Trypsin ◽  
Purification And Properties ◽  
Vipera Ammodytes

A kininogenin (EC 3.4.21.8) was purified from the venom of Vipera ammodytes ammodytes (European sand viper) by a combination of gel filtration and ion-exchange chromatography. The enzyme is approximately six times more active than bovine trypsin in its ability to release vasoactive peptides from a plasma precursor. The kininogenin is a glycoprotein containing 18-20% by weight of carbohydrate. It showed a mol. wt. of 40500 on gel filtration. Gel electrophoresis of the reduced sample in the presence of sodium dodecyl sulphate and 2-mercaptoethanol revealed the presence of two major components of mol.wt. 34300 and 31300. The heterogeneity, which was also observed on disc electrophoresis, was removed by incubation with neuraminidase. After incubation with neuraminidase the kininogenin retained full enzymic activity and possessed an isoelectric point of pH7.2. The carbohydrate content has been decreased to 10% by weight, and the single component seen on electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol corresponded to a mol.wt. of 29500.

scholarly journals Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

1985 ◽  
Vol 231 (2) ◽  
pp. 407-416 ◽  
Author(s):  
N Allison ◽  
M J O'Donnell ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Molecular Masses ◽  
Membrane Bound ◽  
Lactate Dehydrogenases

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

A novel mucin sulphatase from human faeces: its identification, purification and characterization

1992 ◽  
Vol 82 (4) ◽  
pp. 447-454 ◽  
Author(s):  
H. H. Tsai ◽  
D. Sunderland ◽  
G. R. Gibson ◽  
C. A. Hart ◽  
J. M. Rhodes
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Human Faeces ◽  
Colonic Mucin ◽  
Inorganic Sulphate ◽  
Normal Human ◽  
Ph Range

1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3–7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/ kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.

scholarly journals Purification and properties of glutathione peroxidase from human placenta

1979 ◽  
Vol 177 (2) ◽  
pp. 471-476 ◽  
Author(s):  
Y C Awasthi ◽  
D D Dao ◽  
A K Lal ◽  
S K Srivastava
Keyword(s):  
Molecular Weight ◽  
Glutathione Peroxidase ◽  
Human Placenta ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Exchange Chromatography ◽  
Human Erythrocytes ◽  
Kinetic Properties ◽  
Peroxidase Isoenzyme ◽  
Purification And Properties

Glutathione peroxidase (glutathione–H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.

Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A

1977 ◽  
Vol 55 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Peter Hechtman
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolase Activity ◽  
Gm2 Ganglioside ◽  
Heat Stable ◽  
Hexosaminidase A ◽  
Heat Stable Protein ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A fraction.The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates.Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.

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