Purification and properties of an acid proteinase from Choanephora cucurbitarum

1986 ◽  
Vol 32 (2) ◽  
pp. 151-155
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Hydrolytic Activity ◽  
Acid Proteinase ◽  
Metallic Ions ◽  
Diisopropyl Fluorophosphate ◽  
Purification And Properties ◽  
Choanephora Cucurbitarum ◽  
Hydrolysis Of

An acid proteinase has been purified from mycelial extracts of Choanephora cucurbitarum by treatment with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The enzyme hydrolysed haemoglobin rapidly compared with casein, bovine albumin, cytochrome c, and hide powder azure, but failed to hydrolyse any of the synthetic peptides tested. The acid proteinase is a glycoprotein with an apparent molecular weight of 12 700. Optimal hydrolysis of haemoglobin by the proteinase was observed at 20 °C, pH 3.0, and has a Km value of 2.8 mg∙mL−1. Heavy metallic ions, such as Hg2+, Fe2+, and Zn2+, inhibited the hydrolytic activity, whereas Ca2+ and Cu2+ enhanced the enzyme activity by two- and four-fold, respectively, as compared with the controls. Benzamidine and phenylmethanesulfonyl fluoride inhibited severely the enzyme activity, while diisopropyl fluorophosphate, antipain, N-α-p-tosyl-L-lysine chloromethyl ketone inhibited moderately. Phenylmethanesulfonyl fluoride inhibition could be reversed by 2-mercaptoethanol. Reducing agents such as cysteine and dithiothreitol did not enhance the enzyme activity. The data presented suggest that the enzyme has the characteristics of serine proteinases.

Cysteine proteinase of Phascolomyces articulosus: purification and properties

1986 ◽  
Vol 64 (11) ◽  
pp. 2441-2445 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iodoacetic Acid ◽  
Phenylmethylsulfonyl Fluoride ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Salting Out ◽  
Purification And Properties

A proteinase from the mycelial extracts of Phascolomyces articulosus has been purified by salting out with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The proteinase rapidly hydrolysed haemoglobin but failed to hydrolyse any of the synthetic peptides tested. The enzyme is a glycoprotein with an apparent molecular weight of 12 800. The carbohydrate content was estimated to be 65%. It has a temperature optimum of 20 °C, pH optimum of 3.0, and has a Km value of 6.6 mg∙mL−1 for denatured haemoglobin. Iodoacetic acid, iodoacetamide, benzamidine, as well as all the heavy metals tested inhibited the enzyme activity. The enzyme activity was not enhanced by reducing agents such as cysteine, ethylenediaminetetra acetic acid, and dithiothreitol, the latter, however, reversed inhibition by phenylmethylsulfonyl fluoride. The inhibitor studies suggest that the enzyme belongs to the group of cysteine proteinases.

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 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 Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity

1979 ◽  
Vol 177 (1) ◽  
pp. 107-114 ◽  
Author(s):  
T G Villa ◽  
V Notario ◽  
J R Villanueva
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Beta Glucanase ◽  
Hydrolysis Of

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.

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.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

scholarly journals SYNTHESIS OF GLYCOPROTEINS IN A SINGLE IDENTIFIED NEURON OF APLYSIA CALIFORNICA

1974 ◽  
Vol 61 (3) ◽  
pp. 649-664 ◽  
Author(s):  
Richard T. Ambron ◽  
James E. Goldman ◽  
Elizabeth Barnes Thompson ◽  
James H. Schwartz
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Mild Acid Hydrolysis ◽  
Giant Neuron ◽  
Electrophoretic Patterns ◽  
Identified Neuron ◽  
Hydrolysis Of ◽  
Mild Acid

Incorporation of L-[3H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [3H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [3H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [3H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [3H]fucose, gel profiles of particulate fractions labeled with [14C]valine were much more complex.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

scholarly journals Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1973 ◽  
Vol 135 (4) ◽  
pp. 819-826 ◽  
Author(s):  
W. Howard Evans ◽  
Diana O. Hood ◽  
James W. Gurd
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Protein Band ◽  
Phosphodiesterase Activity ◽  
Liver Plasma Membrane ◽  
Purification And Properties ◽  
Nucleotide Pyrophosphatase ◽  
Neutral Sugars

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate–Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose–detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5′-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000–130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD+, UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.

Purification and Properties of a Catechol Methyltransferase of the Yeast Candida tropicalis

1979 ◽  
Vol 34 (9-10) ◽  
pp. 709-714 ◽  
Author(s):  
Joseph Veser ◽  
Peter Geywitz ◽  
Helmut Thomas
Keyword(s):  
Enzyme Activity ◽  
Candida Tropicalis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Methyltransferase Activity ◽  
Purification And Properties ◽  
Mammalian Tissues ◽  
Catechol Methyltransferase ◽  
High Level ◽  
Yeast Fungus

Abstract In an effort to investigate catechol methyltransferase activity in sources other than mammalian tissues and cells, a high level of enzyme activity was found in the yeast fungus Candida tropicalis CBS 94. Partial purification of the enzyme (approx. 550 fold with a recovery of 7%) could be achieved by using ion-exchange and gel filtration techniques. The molecular weight was estimated at 32,000 ± 2,000 by gel filtration on Sephadex G-100. In isoelectric focusing experiments on Sephadex G-75 the enzyme exhibited a pl-value o f 5.0 ± 0.1. In contrast to catechol methyltransferase from various mammalian tissues the enzyme activity was prepared from the pH 5-sediment. The substrate specifity is comparable to other catechol methyltransferases.

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