scholarly journals Purification and properties of myo-inositol-1-phosphatase from bovine brain

1988 ◽  
Vol 253 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P V Attwood ◽  
J B Ducep ◽  
M C Chanal
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Native Enzyme ◽  
Hydroxyl Groups ◽  
Inositol 1 ◽  
Substrate Structure ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Hydrolysis Of

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

scholarly journals The purification and properties of myo-inositol monophosphatase from bovine brain

1988 ◽  
Vol 249 (3) ◽  
pp. 883-889 ◽  
Author(s):  
N S Gee ◽  
C I Ragan ◽  
K J Watling ◽  
S Aspley ◽  
R G Jackson ◽  
...  
Keyword(s):  
Bovine Brain ◽  
Small Extent ◽  
Inositol Monophosphatase ◽  
Inositol 1 ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Hydrolysis Of

1. An inositol monophosphatase was purified to homogeneity from bovine brain. 2. The enzyme is a dimer of subunit Mr 29,000. 3. The enzyme hydrolyses both enantiomers of myo-inositol 1-phosphate and both enantiomers of myo-inositol 4-phosphate, but has no activity towards inositol bisphosphates, inositol trisphosphates or inositol 1,3,4,5-tetrakisphosphate. 4. Several non-inositol-containing monophosphates are also substrates. 5. The enzyme requires Mg2+ for activity, and Zn2+ supports activity to a small extent. 6. Other bivalent cations (including Zn2+) are inhibitors, competitive with Mg2+. 7. Phosphate, but not inositol, is an inhibitor competitive with substrate. 8. Li+ inhibits hydrolysis of inositol 1-phosphate and inositol 4-phosphate uncompetitively with different apparent Ki values (1.0 mM and 0.26 mM respectively).

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

1988 ◽  
Vol 250 (2) ◽  
pp. 453-458 ◽  
Author(s):  
H Sobek ◽  
H Görisch
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Acidocaldarius ◽  
Sedimentation Equilibrium ◽  
Prolonged Storage ◽  
Heat Stable ◽  
Equilibrium Data ◽  
Poly Ethylene ◽  
Hydrolysis Of

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

Production, purification, and properties of glucose oxidase from the biocontrol fungus Talaromyces flavus

1990 ◽  
Vol 36 (3) ◽  
pp. 199-205 ◽  
Author(s):  
Kay K. Kim ◽  
Deborah R. Fravel ◽  
George C. Papavizas
Keyword(s):  
Glucose Oxidase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Native Enzyme ◽  
Purification And Properties ◽  
Talaromyces Flavus ◽  
Optimum Ph ◽  
Extracellular Glucose ◽  
Type Population

Talaromyces flavus produces the enzyme glucose oxidase, which may be involved in biocontrol of the fungal plant pathogen, Verticillium dahliae. A strain of T. flavus was selected from the wild-type population for the production of extracellular glucose oxidase, and the enzyme was purified by a combination of acetone precipitation and high performance liquid chromatography (HPLC). Approximately 12–25 mg of pure protein was obtained from 2 L of culture, and the total recovered activity ranged from 5 to 10 × 103 μmol/min. Homogeneity of the purified enzyme was demonstrated by HPLC and by native and sodium dodecylsulfate polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was 164 000 and that of the subunit was71 000, which indicated that the native enzyme is a dimer. The apparent Km value for D-glucose was 10.9 mM. The optimum pH for the enzyme activity was 5.0, but the enzyme was stable in buffer from pH 3 to 7. The enzyme was observed to be a glycoprotein, and amino acid analysis of the purified enzyme indicated a similarity to glucose oxidases from fungal sources. Isozymes of the enzyme with pI values of 4.40–4.55 were detected on analytical isoelectric focusing gels. Key words: antibiosis, biocontrol, glucose oxidase, Talaromyces flavus.

Phosphoribosyl Pyrophosphate Degradation in Human Tissues

1974 ◽  
Vol 52 (12) ◽  
pp. 1162-1166 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Maximum Activity ◽  
Human Tissues ◽  
Phosphoribosyl Pyrophosphate ◽  
Phosphorylated Compounds ◽  
Tissue Homogenates ◽  
Sugar Derivatives ◽  
Hydrolysis Of ◽  
Alkaline Range

Enzymatic hydrolysis of phosphoribosyl pyrophosphate (PP-ribose-P) in dialyzed human tissue homogenates had a specific activity ranging from 0.15 to 1.37 μmol mg−1 h−1. Our observations on human placenta have characterized this reaction: (1) PP-ribose-P was degraded in the absence of Mg with stoichiometric release of Pi at 37 °C; (2) the reaction occurred from pH 5 to 10.5 with the maximum activity in the alkaline range; (3) PP-ribose-P degrading activity was localized mainly in the microsomal fraction; (4) alkaline phosphatase rather than 5′-nucleotidase was responsible for the degradation of PP-ribose-P; (5) the Km for PP-ribose-P was 0.3 mM; (6) PP-ribose-P hydrolysis was altered by many phosphorylated compounds, both nucleoside and sugar derivatives, Pi, and PPi in concentrations ranging from 0.1 to 10.0 mM.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

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