scholarly journals Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver

1984 ◽  
Vol 220 (3) ◽  
pp. 825-833 ◽  
Author(s):  
S C Butterwith ◽  
R Hopewell ◽  
D N Brindley
Keyword(s):  
Calcium Phosphate ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Thiol Groups ◽  
Purification And Characterization ◽  
Phosphatidate Phosphohydrolase ◽  
Arginine Residues

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

scholarly journals Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7545
Author(s):  
Jianyou Zhang ◽  
Guangcheng Zhou ◽  
Lifeng Fei ◽  
Lifan Chen ◽  
Lei Sun ◽  
...  
Keyword(s):  
Polyphenol Oxidase ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Enzymatic Reactions ◽  
Inhibitory Effects ◽  
Purification And Characterization ◽  
Effective Prevention ◽  
Ammonium Sulphate Precipitation

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals PURIFIKASI PARSIAL DAN KARAKTERISASI ß-GALAKTOSIDASE Lactobacillus plantarum B123 INDIGENOS DAN HIDROLISIS LAKTOSA UNTUK PRODUKSI SUSU ULTRA HIGH TEMPERATURE RENDAH LAKTOSA

2015 ◽  
Vol 17 (2) ◽  
pp. 147-161
Author(s):  
Tatik Khusniati ◽  
Neny Mariyani ◽  
Hanifah Nuryani Lioe ◽  
Didah Nur Faridah ◽  
Abdul Choliq ◽  
...  
Keyword(s):  
High Temperature ◽  
Lactobacillus Plantarum ◽  
Lactose Intolerance ◽  
Specific Activity ◽  
Partial Purification ◽  
Lactose Hydrolysis ◽  
Galactosidase Activity ◽  
Purification And Characterization ◽  
Uht Milk

β-Galactosidase is enzyme which hidrolyze lactose to glucose and galactose. This enzyme is used in production low lactose milk for consumption human which have lactose intolerance. Partial purification of β-galactosidase is important to be conducted to increase  β-galactosidase activity in order to its hydrolysis potency on UHT milk lactose increased.This research was aimed to production by partially purification and characterization indigenous β-galactosidase from Lactobacillus plantarum B123, and lactose hydrolysis for production low lactose UHT milk. Partially purification were precipitation following dialysis. Characterization included optimazion and stabilization of enzyme, while lactose hydrolisis for production low lactose UHT milk was detected by enzymatic GOD-POD kit. The results showed that production of β-galactosidase by using partial purification increased from 21.51 ± 0.23 U/mL (crude) to 106.34 ± 0.56 U/mL (dialysis).  The optimum crude β-galactosidase activity was reached in precipitation by using 60 % ammonium sulphate.  The purity of crude β-galactosidase increased 3.71 times after precipitation, and 14.28  times  after dialysis. Characterization of β-galactosidase showed that  optimum activities of crude and dialyzed β-galactosidase were at pH 6.5 and 50 oC, respectively. Stability of crude β-galactosidase incubated for 1 h were at pH: 5.0-8.5 and 25-50 °C. Specific activity of crude β-galactosidase was 15.05 U/mg protein, while that dialyzed β-galactosidase was 109.58 U/mg protein. Lactose hidrolysis to produce low lactose UHT milk showed that glucose concentration increased with the increase of hidrolysis time. Time needed to hidrolyze lactose 50 % with 4.8 U/mL β-galactosidase at 50°C was 6.08 h. In conclusion that indigenous β-galactosidase from Lactobacillus plantarum B123 purified partially can be used as lactose hidrolyzer in production of low lactose UHT milk.Key words : b-galactosidase, indigenous Lactobacillus plantarum B123, purification, lactose hidrolysis, UHT Milk

scholarly journals Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen

1986 ◽  
Vol 237 (2) ◽  
pp. 439-445 ◽  
Author(s):  
J Gomez-Cambronero ◽  
S Velasco ◽  
M Sanchez-Crespo ◽  
F Vivanco ◽  
J M Mato
Keyword(s):  
Specific Activity ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Acetyl Coa ◽  
Ph Optimum

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.

scholarly journals Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of γ-glutamyl kinase

1979 ◽  
Vol 181 (1) ◽  
pp. 215-222 ◽  
Author(s):  
R V Krishna ◽  
T Leisinger
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinase Activity ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Gamma Glutamyl ◽  
Hydrolysis Of ◽  
Molecular Sieving

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.

scholarly journals Molecular Identification of Indigenous Lactic Acid Bacteria, Partial Purification and Characterization of β-Galactosidase Produced

2017 ◽  
Vol 3 (4) ◽  
pp. 247
Author(s):  
Tatik Khusniati ◽  
Sulistiania . ◽  
Bellen Nastitie Pamela Fury ◽  
Syamsul Falah ◽  
Abdul Choliq
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Ammonium Sulphate ◽  
Molecular Identification ◽  
Specific Activity ◽  
Partial Purification ◽  
Optimum Temperature ◽  
Purification And Characterization

<p>β-Galactosidase is enzyme which hydrolyze lactose to glucose and galactose, as lactose hydrolyzer. To know indigenous lactic acid bacteria (LAB) characteristics, indigenous LAB identification, partial purification and characterization of β-galactosidase produced was researched. LAB was molecularly identified, β-galactosidase partial purification was conducted by precipitation followed dialysis. β-Galactosidase  characterization was based on optimum activities of pH and temperature. The results show that LAB was identified as <em>Lactobacillus plantarum</em> B123. Optimum activity of precipited β-galactosidase was reached at 50 % ammonium sulphate. Activity and specific activity of 50 % precipited β-galactosidase were 95.675 U · mg<sup>–1</sup> and 32.268 U · mg<sup>–1</sup>, respectively. Precipited β-galactosidase resulted a purification level of 3.99 fold, a yield of 38.73 %, and a specific activity of 32.27 U · mg<sup>–1</sup> protein, while dialyzed β-galactosidase resulted 7.61 fold, 10.67 %, and 61.53 U· mg<sup>–1</sup> protein. Optimum temperature and pH for crude β-galactosidase were found at 55 °C and 7.0, respectively, while that dialyzed β-galactosidase were optimized at 50 °C and 7.0. Based on partial purification and characterization, <em>Lactobacillus plantarum</em> B123 is indigenous LAB which good for production of β-galactosidase.</p><div><p class="Els-keywords"><em> </em></p><p class="Els-keywords"><strong>Keywords:</strong> characterization; dialysis; lactic acid bacteria; <em>Lactobacillus plantarum</em> B 123; β-galactosidase.</p></div>

scholarly journals Purification and characterization of a lysosomal form and a variant form of β-glucuronidase from the rat basophil leukaemia tumour

1981 ◽  
Vol 193 (3) ◽  
pp. 663-670 ◽  
Author(s):  
L B Schwartz ◽  
K F Austen
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Rat Tissues ◽  
Variant Form ◽  
Polyacrylamide Gels ◽  
Purification And Characterization ◽  
Normal Rat ◽  
Single Subunit

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A–Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.

Sign in / Sign up

Export Citation Format

Share Document