scholarly journals Purification and properties of rabbit spermatozoal acrosomal neuraminidase

1977 ◽  
Vol 161 (2) ◽  
pp. 193-200 ◽  
Author(s):  
P N Srivastava ◽  
H Abou-Issa
Keyword(s):  
Enzyme Activity ◽  
Sialic Acid ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Submaxillary Gland ◽  
Subsequent Treatment ◽  
Major Band ◽  
Acrosomal Membrane ◽  
Purification And Properties ◽  
Freeze Dried

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6′-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals Purification and Properties of a Glucuronan Lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472)

2001 ◽  
Vol 67 (11) ◽  
pp. 5197-5203 ◽  
Author(s):  
Alexandre Da Costa ◽  
Philippe Michaud ◽  
Emmanuel Petit ◽  
Alain Heyraud ◽  
Philippe Colin-Morel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Sinorhizobium Meliloti ◽  
Specific Activity ◽  
Protein Sequences ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn2+, Cu2+, and Hg2+ (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified fromS. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.

The purification and properties of an amino acid arylamidase from bovine milk

1969 ◽  
Vol 47 (2) ◽  
pp. 173-178 ◽  
Author(s):  
A. Mellors
Keyword(s):  
Amino Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Bovine Milk ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Divalent Metal Ions ◽  
Purification And Properties ◽  
Milk Fat Globule ◽  
High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

scholarly journals Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

2019 ◽  
Vol 1 (4) ◽  
pp. 8-18
Author(s):  
Samir A.M. Zaahkouk ◽  
Doaa A. Darwish ◽  
Hassan M.M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Ovis Aries ◽  
Major Band ◽  
Sheep Liver ◽  
Sds Page ◽  
Commercially Important ◽  
Important Enzyme

Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.

scholarly journals Purification and properties of a β-1,6-glucanase from Penicillium brefeldianum

1984 ◽  
Vol 223 (3) ◽  
pp. 707-714 ◽  
Author(s):  
G P Schep ◽  
M G Shepherd ◽  
P A Sullivan
Keyword(s):  
High Pressure ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Dry Weight ◽  
Protein Band ◽  
Purification And Properties ◽  
Freeze Dried ◽  
Beta Glucosidase

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.

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.

Purification and Properties of Chalcone Synthase from Cell Suspension Cultures of Soybean

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1200-1206 ◽  
Author(s):  
Roland Welle ◽  
Hans Grisebach
Keyword(s):  
Cell Suspension ◽  
Chalcone Synthase ◽  
Specific Activity ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Major Band ◽  
Apparent Homogeneity ◽  
Step Procedure ◽  
Purification And Properties ◽  
Soybean Cell

Incubation of soybean cell suspension cultures in B-5 medium containing 0.4 ᴍ sucrose caused induction of chalcone synthase (CHS) up to a specific activity of 8 µkat/kg. CHS from these cultures was purified to apparent homogeneity by a 5-step procedure. Isoelectric focussing of pure CHS gave a major band at pH 5.45 and two weaker bands at pH 5.35 and 5.5. At least the bands at pH 5.35 and 5.45 had CHS activity. Analysis of pure CHS by two-dimensional electrophoresis gave a set ot six proteins with a M, of 40 kDa and pIs between 6.0 and 6.6. One-dimensional PAGE of CHS under non-denaturing conditions gave three closely spaced protein bands. A specific antibody was raised against soybean CHS which cross-reacted with parsley CHS. Attempts to find synthesis of deoxychalconc or of the corresponding 7.4′-dihydroxyflavanonc with CHS of different purification stages and with various cofactors failed.

Purification and properties of phosphoenolpyruvate carboxylase from green leaves of maize

1979 ◽  
Vol 44 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Jaroslav Mareš ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Chloride Ions ◽  
Magnesium Ions ◽  
Ph Optimum ◽  
Green Leaves ◽  
Purification And Properties ◽  
Fructose 1,6 Bisphosphate ◽  
No Activation

Phosphoenolpyruvate carboxylate was isolated from green leaves of maize (Zea mays L.) by a procedure including fractionation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on polyacrylamide gel. The specific activity of the electrophoretically homogeneous enzyme was 23 U/mg. Its molecular weight was about 405 000, pH optimum was within the range 7.9 to 8.3, Km for phosphoenolpyruvate was 1.05 . 10-3 and the apparent Km for the magnesium ions was 8.0 . 10-4M. The enzyme was inhibited by malate, aspartate, citrate, pyruvate, ATP and ADP and chloride ions. It was strongly activated by glycine and glucose 6-phosphate and to a lesser degree by glucose 1-phosphate and fructose 1,6-bisphosphate; no activation by orthophosphate and 3-phosphoglycerate was observed.

scholarly journals Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes

1985 ◽  
Vol 230 (1) ◽  
pp. 25-34 ◽  
Author(s):  
P N B Gibbs ◽  
A G Chaudhry ◽  
P M Jordan
Keyword(s):  
Enzyme Activity ◽  
Dissociation Constant ◽  
Specific Activity ◽  
Enzymic Activity ◽  
Thiol Groups ◽  
General Application ◽  
Erythrocyte Enzymes ◽  
Human Enzyme ◽  
Purification And Properties ◽  
Porphobilinogen Synthase

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.

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