scholarly journals Purification and some properties of arylsulphatases A and B from rabbit kidney cortex

1977 ◽  
Vol 165 (1) ◽  
pp. 127-134 ◽  
Author(s):  
J J Helwig ◽  
A A Farooqui ◽  
C Bollack ◽  
P Mandel
Keyword(s):  
Ascorbic Acid ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Chromatography Method ◽  
Arylsulphatase A ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N–acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.

scholarly journals Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex

1978 ◽  
Vol 175 (1) ◽  
pp. 321-329 ◽  
Author(s):  
J J Helwig ◽  
A A Farooqui ◽  
C Bollack ◽  
P Mandel
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Hydrolysis Rate ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Basic Form ◽  
Nitrophenyl Phosphate ◽  
Acidic Form

Two forms of tartrate-sensitive acid phosphatases (EC 3.1.3.2) were purified from rabbit kidney cortex by a multiple-column-chromatography method. The basic form constituted 90% of the enzyme and migrated as a single band of protein on polyacrylamide-gel electrophoresis. The proteins contaminating the acidic form did not exceed 5% of the total protein. The specific activity towards p-nitrophenyl phosphate was 12 mumol/min per mg for the basic form and 0.7 mumol/min per mg for the acidic form. The basic form of the enzyme differs from the acidic form in its heat-stability, Km values, inhibition rates by tartrate and fluoride and substrate specificities. Relative to p-nitrophenyl phosphate hydrolysis rate, the acidic form hydrolysed a variety of physiological monophosphate esters, whereas the basic form hydrolysed only CMP and phosphoenolpyruvate. Bacterial neuraminidases had no effect on the activity and mobility of the acidic form on polyacrylamide-gel electrophoresis. Both forms have the same molecular weight (101000 +/- 4000) and are probably composed of two identical subunits. The question whether the two forms of the enzyme are different proteins or whether one is a modified form of the other is discussed.

Determination of the proportion of κ-casein hydrolysed by rennet on coagulation of skim-milk

1980 ◽  
Vol 47 (3) ◽  
pp. 351-358 ◽  
Author(s):  
Brian Chaplin ◽  
Margaret L. Green
Keyword(s):  
Quantitative Determination ◽  
Gel Electrophoresis ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Skim Milk ◽  
Casein Micelle ◽  
Clotting Time ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

SummaryA method has been developed for quantitative determination of para-κ-casein, involving spectrophotometric scanning of stained protein bands following polyacrylamide gel electrophoresis. The rate of hydrolysis of κ-casein in skim-milk at pH 6·6 and 30 °C was compared with that in EDTA-treated skim-milk under the same conditions. This showed that at the visually observed clotting time, at least 90% of the total κ-casein in milk had been hydrolysed. The time course of the reaction was consistent with all the κ-casein molecules being hydrolysed with the same efficiency. The results strongly suggest that essentially all of the κ-casein in milk is equally accessible to rennet action. This is consistent with the casein micelle being porous, or having all the κ-casein on the surface.

Kinetic and electrophoretic studies on acid and alkaline phosphatases of the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

1982 ◽  
Vol 56 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Afzal A. Siddiqui ◽  
Wajih A. Nizami
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Arsenate ◽  
Alkaline Phosphatases ◽  
Acid And Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Clinostomum Complanatum

ABSTRACTHomogenates of the metacercariae of Clinostomum complanatum showed two peaks for the hydrolysis of p-nitrophenyl phosphate at pH 4·5–5·0 and 11·0; specific activity at pH 5·0 was 0·017 µmol p-nitrophenol/mg protein/min and 0·0049 µmol p-nitrophenol/mg protein/min at pH 11·0 at 37°C. Maximal activities of acid and alkaline phosphatases were observed at 50° and 40°C respectively. Both enzymes reached maximal rates after 50 min of incubation. Linearity in enzyme activity was noticed with increasing homogenate concentrations for both enzymes. The Km for p-nitrophenyl phosphate was 1·1 mM with a Vmax of 0·018 units/mg protein for acid phosphatase and 1·6 mM with a Vmax of 0·0052 units/mg protein for alkaline phosphatase. Sodium arsenate and sodium fluoride inhibit and Mg++ and Co++ stimulate both enzymes. Polyacrylamide gel electrophoresis showed one cathodic band each for both the enzymes.

scholarly journals Peroxidases of Selected Fruits and Vegetables and the Possible Use of Ascorbic Acid as an Antioxidant

HortScience ◽  
1993 ◽  
Vol 28 (1) ◽  
pp. 48-50 ◽  
Author(s):  
G. Préstamo ◽  
P. Manzano
Keyword(s):  
Ascorbic Acid ◽  
Solanum Tuberosum ◽  
Gel Electrophoresis ◽  
Fruits And Vegetables ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Green Beans ◽  
Activity Staining ◽  
Peroxidase Isozymes

The various isozymes of peroxidase of a range of vegetables and kiwifruit were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by specific activity staining. Peroxidase isozymes were determined in potato (Solanum tuberosum L.), carrot (Daucus carota L.), tomato (Lycopersicon esculentum Mill.), kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson], cauliflower [Brassica oleracea (Botrytis group)], green beans (Phaseolus vulgaris L.), and horseradish (Armoracia rusticana Gaertn, Mey Scherb.). There was only one isozyme in cauliflower (70 kDa), two in kiwifruit (45-43 kDa), and a range of isozymes (120-36 kDa) in horseradish. Ascorbic acid inhibited peroxidase activity in the extracts.

scholarly journals Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 697-705 ◽  
Author(s):  
A L Latner ◽  
A W Hodson
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 × 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

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 Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

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