scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

scholarly journals Thermal stability of purified superoxide dismutase from liver of commercially valuable fish, Labeo rohita Exposed to Pb+Cr mixture

2021 ◽  
Vol 58 (04) ◽  
pp. 1191-1196
Author(s):  
Huma Naz
Keyword(s):  
Superoxide Dismutase ◽  
Specific Activity ◽  
Labeo Rohita ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Sod Activity ◽  
Exposed Fish ◽  
Ammonium Sulphate Precipitation ◽  
Km Value ◽  
Increasing Temperature

In this experiment, effect of lead (Pb) + chromium (Cr) mixture on superoxide dismutase (SOD) in the liver of Labeo rohita at a concentration of 11.1 mgL-1 was observed. The ammonium sulphate precipitation and ion exchange chromatography techniques were successfully used to purify SOD. After purification, SOD activity of control and Pb+Cr treated fish was noted as 581.00 and 645.45 UmL-1, respectively while the specific activity was 1383.33 and 1613.62 Umg-1, respectively. The fold purification value of SOD was 2.75 and 2.45 for control and stressed fish, respectively. The recovery was calculated as 77.06 and 57.43% for control and stressed fish, respectively. The results of kinetic characterization showed that SOD form control and exposed fish had maximum activity at pH 6.5 and 7.0. Temperature also had a significant effect on activity of SOD. The SOD activity was measured maximum at 30°C for both control and Pb+Cr exposed fish. The Km value of liver SOD for control and Pb+Cr treated L. rohita was calculated as 1.48 and 0.62 mM, respectively. The value of Vmax for SOD from liver of control and Pb+Cr exposed fish was 1000 and 570 U mL-1, respectively. The enthalpy of denaturation (∆H*) for liver SOD from control and Pb+Cr exposed L. rohita was computed as 3.492 and 2.802 KJ mol-1 at 40°C, respectively and these values were dropped off with increasing the temperature until it remains 3.251 and 2.561 KJ mol-1 at 70°C, respectively. The free energy of thermal denaturation (ΔGº) of liver SOD was slightly increased with increasing temperature until 75°C which shows its resistance against heat. The values of ΔGº was observed as 58.03 and 57.95 KJ mol-1 for control and exposed fish at 40°C, respectively while the same was increased upto 62.37 and 62.00 KJ mol-1 at 70°C, respectively. It was concluded from negative value of ΔS* (entropy of inactivation) that the SOD is stable thermodynamically.

scholarly journals PURIFICATION OF β-GLUCOSIDASE PRODUCED FROM Trichoderma viride USING COW DUNG AS CARBON SOURCE

2020 ◽  
Vol 4 (2) ◽  
Author(s):  
S. T. Tyohemba ◽  
S. Aliyu ◽  
N. N. Ndukwe ◽  
G. G. Memi ◽  
U. O. Edem
Keyword(s):  
Specific Activity ◽  
Trichoderma Viride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Broad Peak ◽  
Cow Dung ◽  
Elution Profile ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Peak Fraction

β-glucosidases have characteristics of biotechnological interest and have thus become important industrial enzymes.In this study, β-glucosidase produced by Trichoderma viride from cow dung was subjected to a three step purification process involving ammonium sulphate precipitation, gel filtration by Sephadex G-100 and ion exchange chromatography by DEAE-Sephadex A-25. The elution profile on Sephadex G-100 resulted in a single broad peak (fractions 9-21) which had a yield of 3.7% and a purification fold of 4.29 with a specific activity of 25.70 µmol/min/mg proteins while the elution profile on DEAE-Sephadex A-25 resulted in a single broad peak (fraction 8-14) which had a yield of 2.76% and a purification fold of 22.14 with a specific activity of 132.41µmol/min/mg of protein. The purified enzyme was obtained as a single band and had a molecular mass of 51.8 kDa on SDS-PAGE. This results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

scholarly journals Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes

1980 ◽  
Vol 186 (2) ◽  
pp. 571-579 ◽  
Author(s):  
G L Francis ◽  
F J Ballard
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Glucose 6 Phosphate Dehydrogenase

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte “ghosts” showed slight activity and erythrocyte and reticulocyte “ghosts” were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.

scholarly journals Purification and Characterization of Cellulase from Termite Ametermes eveuncifer (Silverstri) Soldiers

2016 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
B. S. Fagbohunka ◽  
R. E. Okonji ◽  
Ayinla Zainab Adenike
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Percentage Yield ◽  
Termite Soldiers

Cellulase enzyme was purified and characterized from termite soldiers (Ametermes eveuncifer) using 70% ammonium sulphate precipitation, ion exchange chromatography and affinity chromatography. The enzyme isolated had a specific activity of 5.04 U/mg with a percentage yield of 11.7%. The enzyme showed maximum activity at 500C and pH 8. The enzyme was not inhibited by Ba2+ at a concentration of 1mM and Pb2+ at 10 mM concentration but was inhibited by other metal ions at 1 mM and 10 mM concentrations of their salts (NaCl, KCl, MnCl2, and NiCl2),

scholarly journals Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate

1987 ◽  
Vol 246 (2) ◽  
pp. 263-269 ◽  
Author(s):  
P J Casey ◽  
J M Lowenstein
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Rat Skeletal Muscle ◽  
Adenylosuccinate Lyase ◽  
Apparent Homogeneity ◽  
High Concentrations

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at −70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

scholarly journals Effects of Tannin Extract fromGongronema latifoliumLeaves on LipoxygenaseCucumeropsis maniiSeeds

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Sabinus O. O. Eze ◽  
Bennett C. Nwanguma
Keyword(s):  
Ascorbic Acid ◽  
Propyl Gallate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Oxidative Enzymes ◽  
Kinetic Properties ◽  
Tannin Extract ◽  
Ammonium Sulphate Precipitation ◽  
Show Maximum Activity

Lipoxygenase (EC 1.13.11.12) was partially purified from germinated seeds ofCucumeropsis maniito a purification fold of 47.14, enzyme activity recovery of 72.18%, and specific activity of 326.25 units/mg protein, using a three-step process of centrifugation, ammonium sulphate precipitation and gel filtration. Kinetic properties show maximum activity at pH 6.0 and at optimum temperature of 40°C. Inhibitory effects of the extract fromGongronema latifoliumand two other known antioxidants: ascorbic acid and propyl gallate on lipoxygenase fromCucumeropsis maniiwere studied. Result shows presence of inhibition with IC50 of4.2×10−3±0.09×10−3 g/L,4.3×10−2±0.11×10−2 g/L and7.9×10−2±0.11×10−2 g/L for the extract from ascorbic acid and propyl gallate, respectively. The extract when compared to the other antioxidants exhibits a competitive mechanism of inhibition. This tannin extract could be included during food processing as preservative against food deterioration that might be caused by oxidative enzymes such as lipoxygenase.

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