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 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.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Partial Purification and Characterization of S-Adenosyl-ʟ-Methionine: Norreticuline N-Methyltransferases from Berberis Cell Suspension Cultures

1986 ◽  
Vol 41 (1-2) ◽  
pp. 126-134 ◽  
Author(s):  
Chi-Kit Wat ◽  
Paul Steffens ◽  
Meinhart H. Zenk
Keyword(s):  
Cell Suspension ◽  
Cell Suspension Cultures ◽  
Apparent Molecular Weight ◽  
Suspension Cultures ◽  
Partial Purification ◽  
Enzymatic System ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Ph Optimum

Abstract Two new N-methyltransferases (NMT-I and NMT-II) were found to occur in Berberis vulgaris cell suspension cultures. One of these enzymes (NMT-I) was partially purified (100-fold) and characterized. This enzyme is specific for tetrahydrobenzylisoquinoline alkaloids and S-adenosyl-ʟ-methionine serves as the methyl donor. The apparent molecular weight of the enzyme is 68,000. The pH optimum of the enzyme is 7.6, the temperature optimum 35 °C. Apparent KM values for (R)-tetrahydropapaverin as substrate were 0.2 mᴍ and for SAM 0.04 mᴍ. The preparation of the same type of enzyme from B. wilsoniae var. subcaulialata was utilized as an efficient enzymatic system for the synthesis of stereochemically pure (R)-as well as (S)-reticuline labelled with tritium or 14C at the N-CH3 group. Enzymes catalyzing this type of reactions are named S-adenosyl-ʟ-methionine: norreticuline N-methyltransferases.

Separation, Partial Purification and Characterization of a Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato Fruits

1982 ◽  
Vol 37 (3-4) ◽  
pp. 165-173 ◽  
Author(s):  
P. Schreier ◽  
G. Lorenz
Keyword(s):  
Fatty Acid ◽  
Inhibitory Effect ◽  
Fatty Acid Hydroperoxide ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isoelectric Points ◽  
Acid Hydroperoxide ◽  
Membrane Bound

Abstract A membrane-bound enzyme catalysing the cleavage of 13-hydroperoxy-(Z)-9,(E)-11-oc-tadecadienoic acid (13-LHPO) and 13-hydroperoxy-(Z)-9,(E)-11,(Z)-15-octadecadienoic acid (13-LnHPO) to C6-aldehydes was isolated and partially purified from apples and tomatoes. Attempts to employ Ultrogel AcA 34 and AcA 22 in a gel chromatographic purification step were partially frustrated by reaggregation phenomena. However, by using Sepharose CL-4 B an enzyme fraction (MW 200 000 Da) with lipoxygenase and fatty acid hydroperoxide cleaving activity could be separated from a high molecular-weight active eluate. By applying preparative isoelec­ tric focussing to the tomato protein we succeeded in separating the fatty acid cleaving activity from the lipoxygenase, because o f their different isoelectric points of pH 5.8 -6 .1 and pH 5.0, respectively, An 8.4-fold purification of the fatty acid cleaving activity was achieved. A pH-optimum of 5.5 and a Km-value of 2.6 × 10-5 м/1 for the 13-hydroperoxide of linoleic acid were measured. p-Chloromercuribenzoic acid (1 mм) showed significant inhibitory effect on the fatty acid hydroperoxide cleaving enzyme, but no evidence o f inhibition was found with 1 mм H2O2, KCN, DABCO and EDTA or superoxide dismutase (270 U). The maximum amount of fatty acid hydroperoxide decomposition (C6-aldehyde formation) was determined to be 59%.

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 Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599 ◽  
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

Abstract A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

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

Partial Purification and Characterization of Membrane-Associated 3-Hydroxy-3-methylglutaryl-Coenzyme A Lyase from Radish Seedlings

1993 ◽  
Vol 48 (5-6) ◽  
pp. 444-450 ◽  
Author(s):  
Thomas Weber ◽  
Thomas J. Bach
Keyword(s):  
Activation Energy ◽  
Molecular Mass ◽  
Coenzyme A ◽  
Arrhenius Plot ◽  
Linear Part ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Radish Seedlings

Abstract We solubilized from radish membranes and purified by 154-fold 3-hydroxy-3-methyl-glutaryl-CoA lyase (HMGL, EC 4.1.3.4) catalyzing the conversion of 3-hydroxy-3-methyl-glutaryl-(HMG -)CoA into acetyl-CoA and acetoacetate. The apparent molecular mass under non-denaturating conditions is 70 kDa. The enzyme has a broad pH optimum around 8.0 and its activation energy as determined from the linear part of an Arrhenius plot is 137.1 kJ/mol. The Km with respect to (5)-H MG-CoA is 40 μM . The enzyme is extremely unstable and rapidly loses activity even when kept on ice, but retains some activity over several weeks when stored at -80 °C.

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.

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