scholarly journals Purification and Characterization of a Carbonyl Reductase from Human Liver, which is Competent in the Reduction of 6-Pyruvoyl-Tetrahydropterin

Pteridines ◽  
1989 ◽  
Vol 1 (4) ◽  
pp. 189-198 ◽  
Author(s):  
Petra Steinerstauch ◽  
Yoshitomo Sawada ◽  
Walter Leimbacher ◽  
Sandro Ghisla ◽  
Hans-Christoph Curtius
Keyword(s):  
Human Liver ◽  
Specific Activity ◽  
Polyclonal Antibodies ◽  
Potassium Phosphate ◽  
Side Chain ◽  
Purification And Characterization ◽  
Single Polypeptide Chain ◽  
Apparent Homogeneity ◽  
Single Polypeptide

Summary An enzyme which reduces 6-pyruvoyl-tetrahydropterin has been purified to apparent homogeneity from human liver. It consists of a single polypeptide chain with a molecular weight of 35 kDa, has an isoelectric point of 5.9 ± 0.1 and contains no glycosyl residues. The pure enzyme has a specific activity of 450 mU/mg protein at pH 7.0 in 10 mM potassium phosphate buffer. It converts 6-pyruvoyl-tetrahydropterin to 6-lactoyltetrahydropterin by transfer of the pro 4R-hydrogen of NADPH to form the side chain -OH at position C(2') of the substrate. Km values are 1.8 J..lM for 6-pyruvoyl-tetrahydropterin and 5.5 J..lM for NADPH. Polyclonal antibodies raised against the purified enzyme recognize 6-pyruvoyl-tetrahydropterin reductase in Western blot and ELISA but do not cross-react with human sepiapterin reductase. The enzyme appears to be identical with aldose reductase.

scholarly journals Purification and Characterization of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus penicillioidesIsolate and Its Potential for Souse with Detergents

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Imran Ali ◽  
Ali Akbar ◽  
Mohammad Anwar ◽  
Sehanat Prasongsuk ◽  
Pongtharin Lotrakul ◽  
...  
Keyword(s):  
Specific Activity ◽  
Amylase Activity ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Good Potential

An extracellularα-amylase from the obligate halophilicAspergillus penicillioidesTISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1andVmax⁡andKmvalues of 1.05 µmol·min−1·mg−1and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1NaCl. The addition of CaCl2at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as anα-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

scholarly journals (335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1083C-1083
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations ◽  
Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

scholarly journals Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA)

1988 ◽  
Vol 250 (2) ◽  
pp. 501-507 ◽  
Author(s):  
M Valoti ◽  
L Della Corte ◽  
K F Tipton ◽  
G Sgaragli
Keyword(s):  
Specific Activity ◽  
Maximum Velocity ◽  
Purification And Characterization ◽  
Reaction Progress ◽  
Reversible Aggregation ◽  
Aggregation Behaviour ◽  
Apparent Homogeneity ◽  
Specificity Constant ◽  
Km Value

Impure preparations of rat intestinal peroxidase were shown to aggregate at low ionic strengths and to disaggregate at higher values. This aggregation was accompanied by a decrease in specific activity, which could lead to hysteretic behaviour of reaction progress curves. Advantage was taken of this reversible aggregation to obtain a relatively pure extract, which was subsequently purified to apparent homogeneity by affinity chromatography on concanavalin A-Sepharose followed by hydrophobic chromatography. The purified enzyme did not show the ionic-strength-dependent aggregation behaviour, behaving as a monomer of Mr 50,000. The purified enzyme was shown to catalyse the peroxidatic conversion of the commonly used antioxidant 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) to form 3,3′-di-t-butyl-2,2′-dihydroxy-5,5′-dimethoxybiphenyl, with a Km value of 176 microM and a maximum velocity of 8 mumol/min per mg. The specificity constant, kcat./Km, for this substrate was similar to that shown towards the substrate guaiacol.

scholarly journals Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases

1992 ◽  
Vol 288 (2) ◽  
pp. 637-642 ◽  
Author(s):  
N Divecha ◽  
C E L Brooksbank ◽  
R F Irvine
Keyword(s):  
Bovine Brain ◽  
Kinetic Properties ◽  
Type Ii ◽  
Purification And Characterization ◽  
Kinase C ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Single Polypeptide ◽  
Phosphatidylinositol 4

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

scholarly journals Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides

1996 ◽  
Vol 314 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Kai-Fai LEE ◽  
Yen-Chywan LIAW ◽  
Pang-Chui SHAW
Keyword(s):  
Initial Rate ◽  
Specific Activity ◽  
Overlapping Genes ◽  
Purification And Characterization ◽  
Native Molecular Mass ◽  
Apparent Homogeneity ◽  
Molecular Masses ◽  
Chromatographic Media ◽  
Lambda Dna

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103–108]. Here we describe protocols developed to purify polypeptides α and β together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0×105 units per mg of protein. The respective denatured molecular masses of polypeptides α and β were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 μM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

scholarly journals The purification and characterization of wheat-germ agglutinin

1972 ◽  
Vol 129 (4) ◽  
pp. 847-856 ◽  
Author(s):  
Dreania LeVine ◽  
Michael J. Kaplan ◽  
Peter J. Greenaway
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Equilibrium Dialysis ◽  
Cysteic Acid ◽  
Purification And Characterization ◽  
Single Polypeptide Chain ◽  
C Terminus ◽  
Approximate Molecular Weight ◽  
Single Polypeptide

The purification of wheat-germ agglutinin by precipitation with ammonium sulphate and by chromatography on Sephadex G-75, Sepharose–ovomucoid and CM-cellulose is described. This procedure gave agglutinin preparations which were homogeneous on polyacrylamide gels under a variety of conditions. Purified wheat-germ agglutinin formed colourless solutions and was relatively insoluble at neutral pH; maximum solubility in 1mm-tris–HCl buffer, pH7.4, was approx. 1mg/ml. The agglutinin was a glycoprotein containing a single polypeptide chain with an approximate molecular weight of 23000. The N-terminus of the oxidized agglutinin was cysteic acid and the C-terminus was glycine. The amino acid composition showed that the protein was extremely rich in cysteine and cystine; there were 15–17 free SH groups/mol. The absorption maximum for the protein was at 272nm and the molar extinction coefficient at 280nm was 1.09×105 litre·mol-1·cm-1. Equilibrium dialysis indicated that there was only one binding site per molecule for N-acetylglucosamine.

scholarly journals Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver.

1988 ◽  
Vol 263 (5) ◽  
pp. 2536-2542
Author(s):  
M Laniado-Schwartzman ◽  
K L Davis ◽  
J C McGiff ◽  
R D Levere ◽  
N G Abraham
Keyword(s):  
Arachidonic Acid ◽  
Human Liver ◽  
Purification And Characterization ◽  
Cytochrome P 450
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