scholarly journals Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma

1989 ◽  
Vol 264 (3) ◽  
pp. 721-727 ◽  
Author(s):  
T Dauvrin ◽  
D Thinès-Sempoux
Keyword(s):  
Enzyme Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Product Inhibition ◽  
Maximum Activity ◽  
Kinetic Properties ◽  
Transferase Activity ◽  
Purification And Characterization ◽  
Sepharose 4B

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons

1981 ◽  
Vol 195 (1) ◽  
pp. 71-81 ◽  
Author(s):  
G McKay ◽  
P D Shargool
Keyword(s):  
Pisum Sativum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Coomassie Blue ◽  
Km Value ◽  
Acetylglutamate Kinase

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.

scholarly journals Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

1986 ◽  
Vol 234 (1) ◽  
pp. 157-162 ◽  
Author(s):  
N N Dewji ◽  
D R De-Keyzer ◽  
J L Stirling
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Alpha Subunit ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

Purification and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Wheat Bran

2013 ◽  
Vol 641-642 ◽  
pp. 906-909
Author(s):  
Chun Zhi Zhang ◽  
Ming Chen ◽  
Hai Chen Guo ◽  
Guo Ren Zu ◽  
Li Chen
Keyword(s):  
Molecular Weight ◽  
Wheat Bran ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Purification And Characterization ◽  
Enzyme Solution ◽  
Crude Enzyme Solution

The ginsenoside-hydrolyzing β-glucosidase that can converse the major ginsenosides into the minor ginsenosides was isolated from wheat bran, and the enzyme was purified and characterized. The crude enzyme solution extracted from wheat bran could hydrolyse the protopanaxadiol-type ginsenosides such as Rb1, Rc, Rd and Rg3, but could not hydrolyse the protopanaxatriol-type ginsenosides such as Re and Rg2. The enzyme fractionated on the DEAE-Cellulose DE-52 column was purified to one spot in SDS polyacrylamide gel electrophoresis, and the molecular weight of enzyme in the fraction 34, 47, and 61 was approximately 62 kDa, 62 kDa, and 68 kDa, respectively.

Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum

1996 ◽  
Vol 42 (10) ◽  
pp. 1039-1044 ◽  
Author(s):  
Eliane Ferreira Noronha ◽  
Cirano José Ulhoa
Keyword(s):  
Trichoderma Harzianum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isolated Cell

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Km and Vmax values for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1 and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2 and stimulated by cations such as Zn2+ and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.

scholarly journals The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor

1990 ◽  
Vol 265 (3) ◽  
pp. 735-738 ◽  
Author(s):  
P J White ◽  
J Young ◽  
I S Hunter ◽  
H G Nimmo ◽  
J R Coggins
Keyword(s):  
Neurospora Crassa ◽  
Aspergillus Nidulans ◽  
Gel Electrophoresis ◽  
Streptomyces Coelicolor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Purification And Characterization ◽  
Large Oligomer

The enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from Streptomyces coelicolor. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of SDS was 16,000. The native Mr estimated by gel filtration on a Superose 6 column was 209,000, indicating that the enzyme is a large oligomer. The enzyme was found to be extremely thermostable. This stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the quinate-inducible 3-dehydroquinase found in Neurospora crassa and Aspergillus nidulans. This similarity was confirmed by direct N-terminal sequencing.

scholarly journals Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic Endomycopsis fibuligera Using Sago Starch as a Substrate

2018 ◽  
Vol 16 (3) ◽  
pp. 302
Author(s):  
Ahyar Ahmad ◽  
Harningsih Karim
Keyword(s):  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Degree Of Saturation ◽  
Sago Starch ◽  
Purification And Characterization ◽  
Ammonium Sulphate Fractionation ◽  
Crude Extract Enzyme

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

scholarly journals Purification and characterization of three forms of glutathione S-transferase A. A comparative study of the major YaYa-, YbYb- and YcYc-containing glutathione S-transferases

1982 ◽  
Vol 207 (3) ◽  
pp. 459-470 ◽  
Author(s):  
J D Hayes ◽  
G H D Clarkson
Keyword(s):  
Single Gene ◽  
Deae Cellulose ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Glutathione S Transferases ◽  
Peptide Maps

Rat liver glutathione S-transferases have previously been defined by their elution behaviour from DEAE-cellulose and CM-cellulose as M, E, D, C, B, A and AA. These enzymes are dimeric proteins which comprise subunits of mol.wt. 22 000 (Ya), 23 500 (Yb) or 25 000 (Yc). Evidence is presented that YaYa protein, one of two previously described lithocholate-binding proteins which exhibit transferase activity, is an additional enzyme which is not included in the M, E, D, C, B, A and AA nomenclature. We therefore propose that this enzyme is designated transferase YaYa. Transferases YaYa, C, A and AA have molecular weights of 44 000, 47 000, 47 000 and 50 000 respectively and each comprises two subunits of identical size. These enzymes were purified to allow a study of their structural and functional relationships. In addition, transferase A was further resolved into three forms (A1, A2 and A3) which possess identical activities and structures and appear to be the product of a single gene. Transferases YaYa, C, A and AA each had distinct enzymic properties and were inhibited by cholate. The recently proposed proteolytic model, which attributes the presence of multiple forms of glutathione S-transferase activity to partial proteolysis of transferase AA, was tested and shown to be highly improbable. Peptide maps showed significant differences between transferases YaYa, C, A and AA. Immunotitration studies demonstrated that antisera raised against transferases YaYa and C did not precipitate transferase AA.

Purification and Characterization of an S-adenosyI-L-methionine:flavonoid 3'-0-methyltransferase from Leaves of Trillium apetalon Makino

1999 ◽  
Vol 54 (7-8) ◽  
pp. 501-507
Author(s):  
Yuki Nakamura ◽  
Susumu Teramoto ◽  
Kunijiro Yoshitama
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Leaf Extract ◽  
Deae Cellulose ◽  
Flavonoid Biosynthesis ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Group Transfer ◽  
Sepharose 4B

Abstract In the leaf extract of Trillium apetalon (Liliaceae) distributed in Japan, an enzyme was demonstrated which catalyzes a m ethyl group transfer from S-adenosyl-ʟ-m ethionine (SAM) to the 3′ position of quercetin and its glycosides. The enzyme ( Trillium F3′OMT ) was purified 433-fold with a yield of 0.2% by (NH4)2SO4 precipitation and chromatographies of DEAE - cellulose, SAH-EAH-Sepharose 4B , Sephacryl S-200 and additional chrom atofocusing. Trillium F3′OMT has a pH optimum of 7.0 and a pi of 5.3. The apparent m olecular weight was estimated by Sephacryl S-200 to be about 78 kD a; SD S-PAGE profile showed that the enzyme was a dim er com posed of MW 38 kDa 2 subunits. The enzyme activity was stimulated by EDTA and dithiothreitol (DTT), but strongly inhibited by p-chlorom ercuribenzoate (PCMB) and iodoacetate. The activity was m oderately inhibited by Mg2+ and Zn2+, and strongly inhibited by Co2+, Mn2+ and Hg2+. The apparent Km values for quercetin and SAM were 10 μm and 3.6 μm , respectively. Lower substrate specificity of the glycosides compared with quercetin indicates that methylation precedes glycosylation in flavonoid biosynthesis of T. apetalon

scholarly journals Purification and characterization of ribonucleases from human seminal plasma

1983 ◽  
Vol 215 (3) ◽  
pp. 605-612 ◽  
Author(s):  
C L Lee ◽  
S S L Li ◽  
C Y Li ◽  
T M Chu
Keyword(s):  
Seminal Plasma ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Affinity Column ◽  
Human Seminal Plasma ◽  
Immunological Reactions ◽  
Sepharose 4B

Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5′-(4-aminophenylphospho)uridine 2′(3′)-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.

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