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.

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.

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 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 Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside

1980 ◽  
Vol 185 (3) ◽  
pp. 583-591 ◽  
Author(s):  
Peter Hechtman ◽  
Zarin Kachra
Keyword(s):  
Human Liver ◽  
Anionic Surfactants ◽  
Sodium Taurocholate ◽  
Ionic Surfactants ◽  
Enzymic Hydrolysis ◽  
Gm2 Ganglioside ◽  
Activator Protein ◽  
Hexosaminidase A ◽  
Triton X ◽  
Hydrolysis Of

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of Gm2 ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315–324; Hechtman & Leblanc (1977) Biochem. J.167, 693–701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of Gm2 ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-β-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of Gm2 ganglioside decrease the Km for Gm2 ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and Gm2 ganglioside was obtained by comparing gel-filtration profiles of 3H-labelled GM2 ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and GM2 ganglioside is the preferred substrate for enzymic hydrolysis.

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 properties of human kidney-cortex hexosaminidases A and B

1977 ◽  
Vol 165 (1) ◽  
pp. 49-53 ◽  
Author(s):  
J E Wiktorowicz ◽  
Y C Awasthi ◽  
A Kurosky ◽  
S K Srivastava
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kidney Cortex ◽  
Enzyme Replacement ◽  
Purification And Properties ◽  
Hexosaminidase A ◽  
Specific Activities

Hexosaminidases (EC 3.2.1.30) A and B from human kidney cortex were purified to homogeneity by using concanavalin A affinity chromatography, ion-exchange chromatography and gel filtration. The yield of homogeneous isoenzymes improved approx. 20-fold, giving preparations of hexosaminidases A and B with specific activities of about 200 and 325 units/mg of protein respectively. The kinetic and structural properties of kidney hexosaminidase isoenzymes were studied and compared with the hexosaminidase isoenzymes from human placenta. The amino acid composition of hexosaminidase A was significantly different from that of hexosaminidase B. In the event of success in developing enzyme-replacement therapy for Tay-Sachs and Sandhoff's diseases, this modified procedure can furnish larger amounts of homogeneous isoenzymes.

Identification of N-Monoacetylcystine in Uraemic Plasma

1981 ◽  
Vol 60 (3) ◽  
pp. 331-334 ◽  
Author(s):  
F. Gejyo ◽  
G. Ito ◽  
Y. Kinoshita
Keyword(s):  
Plasma Levels ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Cysteic Acid ◽  
Homocysteic Acid ◽  
Normal Limits ◽  
Acidic Nature ◽  
Sulphur Amino Acids

1. An unidentified ninhydrin-positive substance of an acidic nature was detected in the plasma of uraemic patients. This substance was isolated from haemodialysate by ion-exchange chromatography and gel filtration, and identified as a sulphur-containing amino acid: N-monoacetylcystine. 2. The quantitative determination of sulphur amino acids in plasma revealed that the plasma levels of cysteic acid, homocysteic acid, taurine, cystine and cystathionine as well as N-monoacetylcystine in uraemic patients were markedly higher than in normal subjects (P < 0.001 for each). However, the plasma levels of methionine in uraemic patients were within normal limits.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

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.

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