scholarly journals Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum

1975 ◽  
Vol 147 (2) ◽  
pp. 267-274 ◽  
Author(s):  
A Higa ◽  
J J Cazzulo
Keyword(s):  
Salt Concentration ◽  
Citrate Synthase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Extreme Halophile ◽  
Enzyme Molecule ◽  
Acetyl Coa ◽  
High Concentrations

1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

1983 ◽  
Vol 50 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Patrick F. Fox ◽  
Leszek Stepaniak
Keyword(s):  
Phosphate Buffer ◽  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Lipase Production ◽  
Maximum Activity ◽  
Temperature Range ◽  
Milk Serum ◽  
Heat Stable ◽  
Ph Range

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

The purification and properties of an amino acid arylamidase from bovine milk

1969 ◽  
Vol 47 (2) ◽  
pp. 173-178 ◽  
Author(s):  
A. Mellors
Keyword(s):  
Amino Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Bovine Milk ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Divalent Metal Ions ◽  
Purification And Properties ◽  
Milk Fat Globule ◽  
High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

scholarly journals β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism

1973 ◽  
Vol 134 (1) ◽  
pp. 239-248 ◽  
Author(s):  
Volker Oeding ◽  
Hans G. Schlegel
Keyword(s):  
Condensation Reaction ◽  
Gradient Centrifugation ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Saturation Curve ◽  
Cleavage Reaction ◽  
Acetyl Coa ◽  
Freeze Dried ◽  
Exclusion Chromatography

1. β-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000–150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris–HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The Km was 3.9×10−4m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the β-ketothiolase condensation reaction and its possible involvement in the regulation of poly-β-hydroxybutyrate synthesis and degradation are discussed.

scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

scholarly journals Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme

1982 ◽  
Vol 205 (1) ◽  
pp. 69-74 ◽  
Author(s):  
E W Gold
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Umbilical Cord ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Significant Difference ◽  
High Concentrations ◽  
Different Response

Human liver hyaluronidase was purified to homogeneity by (NH4)2SO4 fractionation, chromatography on hydroxyapatite and DEAE-cellulose, and preparative disc polyacrylamide-gel electrophoresis. The enzyme had a pH optimum of 3.8-4.0, a molecular weight (determined by gel filtration) of 76000, and a Km of 0.05 mg/ml for purified human umbilical-cord hyaluronic acid. It generally resembled hyaluronidases studied in other tissues which are believed to be lysosomal, but shared a number of characteristics with a partially purified bovine testicular hyaluronidase. Neither enzyme exhibited inhibition by high concentrations of substrate, but both were competitively inhibited by dermatan sulphate and keratan sulphate. Both enzymes exhibited increased activity in the presence of albumin, probably owing to an increased susceptibility of substrate to enzyme action. The liver enzyme was inhibited by NaCl, but the testicular enzyme exhibited an increase in activity in the presence of the salt which was similar to the effect observed with albumin. The different response toward Cl- ion appeared to be the most significant difference between the two enzymes.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

scholarly journals Isolation, Purification, And Characterization Of Some Cystein Proteases From Bovine Mastites Milk

2009 ◽  
Vol 3 (1) ◽  
pp. 85-97
Author(s):  
K.S. Doosh
Keyword(s):  
Cathepsin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Mastitis ◽  
Deae Cellulose ◽  
Cysteine Proteases ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Molecular Weights

Proteolytic activity of cysteine proteases were studied in bovine mastitis milk, four fractions designated as F1,F2,F3,F4 with cysteine protease activity were separated from leukocytes cell by ion- exchange Chromatography through DEAE-Cellulose The most active fraction F4 was selected for further purification utilizing gel filtration Chromatography on Sephadex G-100 column it has been found that F4 most likely being cathepsin B. purification folds and the enzyme yield was 46.66 and 31.81% respectively . polyacrylamide gel electrophoresis test indicated that the enzyme has been purified to homogeneity by giving a single band . The results of enzyme characterization showed that the molecular weights were 31000 and 30000 Daltons as determined by gel filtration and electrophoresis methods in present of reducing agent SDS- PAGE respectively. The optimum pH for the enzyme activity was 6.0 and it was stable at pH values ranged between 4.5 - 6.5. The enzyme exhibited the maximum activity at 45ºC and the enzyme retained its entire activity over 30 min incubation at 30 -50 C and it retained (50, 20, 10) % of its entire activites over 30 min incubation at (60, 70, 80) C respectively. From this results and results observed from the effect of inhibitor and activator reagents we suggest that enzyme F4 possibly belonged to cathepsin B.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

Sign in / Sign up

Export Citation Format

Share Document