scholarly journals Purification and Properties of Human Erythrocyte Arginase

1989 ◽  
Vol 26 (6) ◽  
pp. 547-553 ◽  
Author(s):  
Masaki Ikemoto ◽  
Masayoshi Tabata ◽  
Takashi Murachi ◽  
Masayuki Totani
Keyword(s):  
Human Erythrocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Immunoaffinity Chromatography ◽  
Acetone Powder ◽  
Purification And Properties ◽  
New Procedures ◽  
Powder Extract ◽  
Specific Polyclonal Antibody

An efficient method for purification of human erythrocyte arginase was developed. This method included two new procedures, hydrophobic chromatography and immunoaffinity chromatography, and yielded 0·7 mg of homogeneous arginase protein from 2·1 L of haemolysate. The molecular weight of native arginase was estimated to be 105 000 by gel filtration on a Sephadex G-150 column, and that of its subunit 35 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This indicates that the native enzyme is composed of three homologous subunits. Amino acid composition of human erythrocyte arginase was found to be very similar to that of liver arginase of several other mammals. After dialysis against distilled water, the purified arginase still retained its enzymatic activity which was decreased by EDTA and reversibly restored by Mn(II) ion. A specific polyclonal antibody for use in an immunoassay was also produced. This antibody revealed one single band on immunoelectrophoretic analysis of the acetone powder extract, suggesting absence of arginase isoenzymes in human erythrocytes.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

Further purification and properties of rat uterine peroxidase

1981 ◽  
Vol 59 (11-12) ◽  
pp. 916-920 ◽  
Author(s):  
Hugh S. Keeping ◽  
Shioko Kimura ◽  
Jane Lovsted ◽  
Peter H. Jellinck
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Prolonged Storage ◽  
High Recovery ◽  
Partial Protection ◽  
Purification And Properties ◽  
Single Protein ◽  
Protein Molecular Weight

Peroxidase was purified 3700-fold from homogenates of estradiol-treated rat uteri by affinity chromatography on concanavalin A (ConA) – Sepharose followed by gel filtration on Bio-Gel P-150 with high recovery of enzyme. A single protein (molecular weight (MW) 45 000) staining for heme was shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis to be present in the peak fractions of enzymic activity eluted from the ConA–Sepharose column. This protein had the same mobility as bovine lactoperoxidase (MW 78 000) in a cationic gel electrophoretic system under nondenaturing conditions. Peroxidase activity in a NaCl extract of the uterus was lower than that in a CaCl2 extract but was unaffected by prolonged storage at −20 °C. In contrast, the CaCl2-extracted enzyme lost much, of its activity under these conditions by a process which could be prevented by the addition of glycerol. The sulfhydryl reagent, N-ethylmaleimide, which caused a marked increase in the activity of uterine peroxidase, provided only partial protection against inactivation during storage of CaCl2 extracts of this enzyme at low temperature.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis

1985 ◽  
Vol 232 (1) ◽  
pp. 151-160 ◽  
Author(s):  
G J Hart ◽  
A R Battersby
Keyword(s):  
Euglena Gracilis ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tyrosine Residues ◽  
Purification And Properties ◽  
Lysine Residues ◽  
Arginine Residues

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.

Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp.

1992 ◽  
Vol 38 (5) ◽  
pp. 436-442 ◽  
Author(s):  
Devyani Dey ◽  
Jyoti Hinge ◽  
Abhay Shendye ◽  
Mala Rao
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Bacillus Sp ◽  
Thermophilic Bacillus ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Similar Temperature

An alkalophilic thermophilic Bacillus sp. (NCIM 59) isolated from soil produced two types of cellulase-free xylanase at pH 10 and 50 °C. The two enzymes (xylanase I and II) were purified to homogeneity by ethanol precipitation followed by Bio-Gel P-10 gel filtration and preparative polyacrylamide gel electrophoresis. The molecular weights of xylanase I and II were estimated to be 35 000 and 15 800, respectively, by sodium dodecyl sulfate gel electrophoresis. The enzymes exhibited immunological cross-reactivity and were glycoproteins. They had similar temperature (50–60 °C) and pH (6) optima. Both xylanases were stable at 50 °C at pH 7 for 4 days. However, xylanase I was comparatively more stable than xylanase II at 60 °C. The isoelectric points of xylanase I and II were 4 and 8, respectively. The apparent Km values, using xylan as substrate, were 1.58 and 3.5 mg/mL, and Vmax values were 0.0172 and 0.742 μmol∙min−1∙mg−1, respectively. Both xylanases were inhibited by N-bromosuccinimide, suggesting the involvement of tryptophan in the active site. The hydrolysis patterns demonstrated that the xylanases were endoenzymes. Xylanase I and II yielded mainly xylobiose, xylotriose, and higher xylooligosaccharides, with traces of xylose from xylan. Key words: cellulase-free xylanase, alkalophilic thermophilic Bacillus sp., enzyme purification, characterization.

scholarly journals Purification and Properties of an Esterase from the YeastSaccharomyces cerevisiae and Identification of the Encoding Gene

1999 ◽  
Vol 65 (8) ◽  
pp. 3470-3472 ◽  
Author(s):  
Giuliano Degrassi ◽  
Lasse Uotila ◽  
Raffaella Klima ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Encoding Gene ◽  
Esterase Activities ◽  
Null Mutants

ABSTRACT We purified an intracellular esterase that can function as anS-formylglutathione hydrolase from the yeastSaccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized toS-formylglutathione by S. cerevisiae.

scholarly journals Purification and properties of pantohenase from Pseudomonas fluorescens

1976 ◽  
Vol 157 (2) ◽  
pp. 409-413 ◽  
Author(s):  
R K Airas ◽  
E A Hietanen ◽  
V T Nurmikko
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Fluorescens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Purification And Properties

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.

scholarly journals Purification and properties of an alginate lyase from a marine bacterium

1976 ◽  
Vol 159 (3) ◽  
pp. 707-713 ◽  
Author(s):  
I W Davidson ◽  
I W Sutherland ◽  
C J Lawson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alginate Lyase ◽  
Kinetic Studies ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Bacterial Cells ◽  
Purification And Properties ◽  
Primary Composition

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.

scholarly journals Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1978 ◽  
Vol 175 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Carol M. Blackwell ◽  
John M. Turner
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Coenzyme B12 ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Protein Components ◽  
Inhibited Enzyme

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.

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