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.

scholarly journals Purification and characterization of prostaglandin-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli

1996 ◽  
Vol 313 (1) ◽  
pp. 223-227 ◽  
Author(s):  
David J. MEYER ◽  
Richmond MUIMO ◽  
Michael THOMAS ◽  
David COATES ◽  
R. Elwyn ISAAC
Keyword(s):  
Parasitic Nematode ◽  
Helminth Parasites ◽  
Ascaridia Galli ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Parasitic Helminths ◽  
Accessory Cells ◽  
Glutathione S Transferases ◽  
Prostaglandin H

Comparison of partial primary sequences of sigma-class glutathione S-transferases (GSH) of parasitic helminths and a GSH-dependent prostaglandin (PG)-H D-isomerase of rat immune accessory cells suggested that some of the helminth enzymes may also be involved in PG biosynthesis [Meyer and Thomas (1995) Biochem. J. 311, 739-742]. A soluble GSH transferase of the parasitic nematode Ascaridia galli has now been purified which shows high activity and specificity in the GSH-dependent isomerization of PGH to PGE, comparable to that of the rat spleen enzyme in its isomerization of PGH to PGD, and similarly stimulates the activity of prostaglandin H synthase. The enzyme subunit is structurally related to the rat spleen enzyme and sigma-class GSH transferases of helminths according to the partial primary sequence. The data support the hypothesis that some sigma-class GSH transferases of helminth parasites are involved in PG biosynthesis which, in the case of PGE, is likely to be associated with the subversion or suppression of host immunity. A PG-H E-isomerase of comparable specificity and activity has not previously been isolated.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

scholarly journals Differential induction of class Alpha glutathione S-transferases in mouse liver by the anticarcinogenic antioxidant butylated hydroxyanisole. Purification and characterization of glutathione S-transferase Ya1Ya1

1989 ◽  
Vol 263 (2) ◽  
pp. 393-402 ◽  
Author(s):  
L I McLellan ◽  
J D Hayes
Keyword(s):  
Female Mouse ◽  
Mouse Liver ◽  
Normal Mouse ◽  
Butylated Hydroxyanisole ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Glutathione S Transferases ◽  
Differential Induction ◽  
Carcinogenic Compound

A novel cytosolic Alpha class glutathione S-transferase (GST) that is not normally expressed in mouse liver was found to be markedly induced (at least 20-fold) by the anti-carcinogenic compound butylated hydroxyanisole. This enzyme (designated GST Ya1 Ya1) did not bind to either the S-hexylglutathione-Sepharose or the glutathione-Sepharose affinity matrices, and purification was achieved by using bromosulphophthalein-glutathione-Sepharose. The purified isoenzyme, which comprises subunits of Mr 25,600, was characterized, and its catalytic, electrophoretic, immunochemical and structural properties are documented. GST Ya1 Ya1 was shown to be distinct from the Alpha class GST that is expressed in normal mouse liver and is composed of 25,800-Mr subunits; the Alpha class isoenzyme that is constitutively expressed in the liver is now designated GST Ya3 Ya3. Hepatic concentrations of GST Ya3 Ya3 were not significantly affected when mice were treated with butylated hydroxyanisole. Both Pi class GST (subunit Mr 24,800) and Mu class GST (subunit Mr 26,400) from female mouse liver were induced by dietary butylated hydroxyanisole. By contrast, hepatic concentrations of microsomal GST (subunit Mr 17,300) were unaffected.

scholarly journals Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver

1986 ◽  
Vol 233 (3) ◽  
pp. 789-798 ◽  
Author(s):  
J D Hayes
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Structural Data ◽  
Glutathione S Transferase ◽  
Purification Scheme ◽  
Peptide Maps ◽  
Limited Sequence

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].

scholarly journals Purification and characterization of hemocyte phenoloxidases in Chilo suppressalis walker (Lepidoptera: Crambidae)

2015 ◽  
Vol 67 (4) ◽  
pp. 1119-1125 ◽  
Author(s):  
Seyyedeh Mirhaghparast ◽  
Arash Zibaee ◽  
Hassan Hoda ◽  
Mahmoud Fazeli-Dinan
Keyword(s):  
Kinetic Parameters ◽  
Column Chromatography ◽  
Deae Cellulose ◽  
Chilo Suppressalis ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Specific Activities ◽  
Fast Flow ◽  
Statistical Effects

In the current study, two phenoloxidases (POs) from the larvae of Chilo suppressalis Walker were extracted and purified by column chromatography using Sepharyl G-100 and DEAE-Cellulose fast flow column. Two proteins possessing PO activity, named as POI and POII, were extracted by purification, 5.08- and 5.62-fold, respectively, with 8.94% and 7.31% recoveries, respectively. Also, the specific activities of POI and POII were 0.478 and 0.529 U/mg protein, respectively. Finally, the molecular weights of POI and POII were calculated as 94.6 and 95.7 kDa, respectively. Kinetic parameters of the purified phenoloxidases by Lineweaver-Burk analysis were Vmax of 2.27 and 1.11 U/mg protein and Km of 15.51 and 17.31 mM for POI and POII, respectively. Mg2+ and Cu2+ significantly increased the PO activities. Ca2+ decreased the activity of POI and showed no statistical effects on POII activity. EDTA and DTC significantly inhibited the activities of the purified enzymes, while triethylenetetramine hexaacetic acid (TTHA) and RGTA showed no significant effects on enzymatic activities.

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).

scholarly journals Purification and characterization of the cuticle-degrading proteases produced by an isolate of Beauveria bassiana using the cuticle of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae)

2015 ◽  
Vol 55 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Hossein Firouzbakht ◽  
Arash Zibaee ◽  
Hassan Hoda ◽  
Mohammad Mehdi Sohani
Keyword(s):  
Beauveria Bassiana ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Purification And Characterization ◽  
Fungal Virulence ◽  
Molecular Weights ◽  
Proteolytic Activities ◽  
Andrallus Spinidens ◽  
Fast Flow

Abstract The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel. Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.

scholarly journals Purification and characterization of glutathione S-transferases of human kidney

1987 ◽  
Vol 246 (1) ◽  
pp. 179-186 ◽  
Author(s):  
S V Singh ◽  
T Leal ◽  
G A Ansari ◽  
Y C Awasthi
Keyword(s):  
Functional Properties ◽  
Human Kidney ◽  
Isoenzyme Pattern ◽  
Human Tissues ◽  
Gamma Delta ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Individual Variations ◽  
Glutathione S Transferases

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (α, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST α, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.

Purification and characterization of glutathione S-transferases in human retina

1984 ◽  
Vol 3 (11) ◽  
pp. 1273-1280 ◽  
Author(s):  
Shivendra V. Singh ◽  
Dat D. Dao ◽  
Satish K. Srivastava ◽  
Yogesh C. Awasthi
Keyword(s):  
Human Retina ◽  
Purification And Characterization ◽  
Glutathione S Transferases
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