scholarly journals Inositol Phosphate Phosphatases of Microbiological Origin. Some Properties of a Partially Purified Bacterial (Pseudomonas Sp.) Phytase

1971 ◽  
Vol 24 (3) ◽  
pp. 547 ◽  
Author(s):  
GCJ Irving ◽  
DJ Cosgrove
Keyword(s):  
Ammonium Sulphate ◽  
Inositol Phosphate ◽  
Gel Filtration ◽  
Pseudomonas Sp ◽  
Ammonium Sulphate Fractionation

A phytase (E.C. 3.1.3.8) was extracted from a bacterium (Pseudomonas sp.) and purified 25-fold by ammonium sulphate fractionation, gel filtration, and ionexchange cellulose chromatography. Its properties are compared with the published properties of some plant phytases and a bacterial phytase.

scholarly journals Isopentenyl pyrophosphate isomerase from liver

1968 ◽  
Vol 106 (4) ◽  
pp. 835-840 ◽  
Author(s):  
P. W. Holloway ◽  
G. Popják
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Pig Liver ◽  
Isopentenyl Pyrophosphate ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Dimethylallyl Pyrophosphate

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6·0 and is activated by Mn2+ in preference to Mg2+. The Km value for isopentenyl pyrophosphate is 4×10−6m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.

Large-scale isolation of fraction 1 leaf protein (18S) from lucerne (Medicago sativa L)

1976 ◽  
Vol 86 (3) ◽  
pp. 495-501 ◽  
Author(s):  
W. T. Jones ◽  
J. L. Mangan
Keyword(s):  
Molecular Weight ◽  
Medicago Sativa ◽  
Ammonium Sulphate ◽  
Large Scale ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Fraction 1 Protein ◽  
Ammonium Sulphate Fractionation ◽  
Medicago Sativa L ◽  
Soluble State

SummaryFraction 1 protein (18S) can be isolated in large quantities (order 100 g) in a soluble state by heating lucerne juice, adjusted to pH 6·7 to 6·9, from a Pirie extractor to 63 °C for 10 min. Low speed oentrifugation (2500 g) removed coagulated chloroplast fragments and most of the heat-denatured Fraction 2 proteins. Fraction 1 (18S) protein (> 95% pure) was purified from low molecular weight materials (sugars, phenolics etc.) by ammonium sulphate fractionation and gel filtration on Sephadex G-75 gels.

scholarly journals Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

1969 ◽  
Vol 22 (2) ◽  
pp. 463 ◽  
Author(s):  
KE Moon ◽  
EOP Thompson
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Fraction I Protein ◽  
Ammonium Sulphate Fractionation ◽  
Fraction I ◽  
Ribulose Diphosphate Carboxylase

Fraction I protein isolated from spinach beet chloroplasts was purified by ammonium sulphate fractionation and Sephadex G�200 gel filtration. The isolated protein was reduced and S-carboxymethylated, and the dissociated protein resolved into two distinct subunits by gel filtration on Sephadex G-200 in an 8M urea buffer at pH 10�0.

scholarly journals Purification and properties of sheep liver phosphofructokinase

1969 ◽  
Vol 113 (2) ◽  
pp. 235-242 ◽  
Author(s):  
D. J. H. Brock
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Optimum Conditions ◽  
Sheep Liver ◽  
A Value ◽  
Purification And Properties ◽  
Ammonium Sulphate Fractionation ◽  
Citrate Inhibition

1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4–7μmoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18·5μmoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

Bovine milk esterases

1971 ◽  
Vol 38 (2) ◽  
pp. 171-177 ◽  
Author(s):  
B. J. Kitchen
Keyword(s):  
Ammonium Sulphate ◽  
Bovine Milk ◽  
Skim Milk ◽  
Histochemical Staining ◽  
Major Type ◽  
Polyacrylamide Gels ◽  
Selective Inhibitors ◽  
Choline Ester ◽  
Ultra Filtration ◽  
Ammonium Sulphate Fractionation

SummaryThe type and distribution of esterases in milk has been investigated using selective inhibitors during normal assay procedures and during histochemical staining of polyacrylamide gels. Enzyme solutions were obtained from skim-milk by acid and alkali precipitation, followed by ammonium sulphate fractionation, ultra-filtration and Sephadex G-100 chromatography. The major type of esterase present was an aryl-esterase (E.C. 5.1.1.2) while a smaller amount of a choline-ester hydrolase (E.C. 3.1.1.7; 3.1.1.8) was detected. The significance of these findings is discussed.

scholarly journals Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

2017 ◽  
Vol 7 (4) ◽  
pp. 1 ◽  
Author(s):  
Sreedevi Basavaraju ◽  
Chandrasekhar Kathera ◽  
Pramoda Kumari Jasti
Keyword(s):  
Bacillus Cereus ◽  
Ammonium Sulphate ◽  
Alkaline Protease ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Tween 20 ◽  
Original Activity ◽  
Protease Enzyme

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

GLYCYL-RNA SYNTHETASE OF RAT LIVER: PARTIAL PURIFICATION AND EFFECTS OF SOME METAL IONS ON ITS ACTIVITY

1963 ◽  
Vol 41 (1) ◽  
pp. 1123-1133 ◽  
Author(s):  
M. J. Fraser
Keyword(s):  
Heat Treatment ◽  
Metal Ions ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Partial Purification ◽  
Ammonium Sulphate Fractionation ◽  
Stimulation Of

Glycyl-RNA synthetase has been purified 40-fold from a 105,000 × g supernatant of an homogenate of rat liver by successive precipitation at pH = 5.0, heat treatment at 55 °C for 3.0 minutes in the presence of 1.0 mM ATP, and ammonium sulphate fractionation. The purified fractions catalyzed glycine-dependent ATP-32PP exchange. The effect of some metal ions on glycine activation was studied. Activation occurred in the presence of either Mg++or Mn++. The apparent stimulation of glycine activation by Co++was found to be an artifact.

scholarly journals Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken

1969 ◽  
Vol 115 (4) ◽  
pp. 849-856 ◽  
Author(s):  
F. G. Nóbrega ◽  
P. S. Araujo ◽  
M. Pasetto ◽  
I. Raw
Keyword(s):  
Amino Acids ◽  
Spectral Properties ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gradient Elution ◽  
Closely Related Species ◽  
Ammonium Sulphate Fractionation ◽  
Terminal Amino

1. Cytochrome b5 was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b5 were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ‘fingerprint’ patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.

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