Isoenzymes of glutathione transferase in rat kidney cytosol

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a ‘new’ form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

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Cloning and heterologous expression of cDNA encoding class Alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic α,β-unsaturated carbonyl compounds

Biochemical Journal ◽

10.1042/bj2840313 ◽

1992 ◽

Vol 284 (2) ◽

pp. 313-319 ◽

Cited By ~ 56

Author(s):

G Stenberg ◽

M Ridderström ◽

Å Engström ◽

S E Pemble ◽

B Mannervik

Keyword(s):

Catalytic Activity ◽

Cdna Clone ◽

Carbonyl Compounds ◽

Glutathione Transferase ◽

Ethacrynic Acid ◽

Glutathione Transferases ◽

High Catalytic Activity ◽

Reading Frame ◽

Dna Fragment ◽

Polymerase Chain

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha, beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

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Purification and some properties of anionic glutathione transferases from rat liver cytosol

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)59378-7 ◽

1986 ◽

Vol 40 ◽

pp. 211

Author(s):

Eibai Lee ◽

Shiroh Okuno ◽

Kimio Kariya

Keyword(s):

Rat Liver ◽

Glutathione Transferases ◽

Liver Cytosol ◽

Rat Liver Cytosol

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Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1

Biochemical Journal ◽

10.1042/bj3020383 ◽

1994 ◽

Vol 302 (2) ◽

pp. 383-390 ◽

Cited By ~ 9

Author(s):

R J Cooke ◽

R Björnestedt ◽

K T Douglas ◽

J H McKie ◽

M D King ◽

...

Keyword(s):

Rat Liver ◽

Active Site ◽

Glutathione Transferase ◽

Competitive Inhibitor ◽

Glutathione Transferases ◽

Molecular Graphics ◽

Hydrophobic Region ◽

Preparative Scale ◽

Photoaffinity Labelling ◽

Wide Range

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

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Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases

Biochemical Journal ◽

10.1042/bj2610531 ◽

1989 ◽

Vol 261 (2) ◽

pp. 531-539 ◽

Cited By ~ 23

Author(s):

P Alin ◽

H Jensson ◽

E Cederlund ◽

H Jörnvall ◽

B Mannervik

Keyword(s):

Rat Liver ◽

Glutathione Transferase ◽

Glutathione Transferases ◽

Rat Lung ◽

Cytosol Fraction ◽

Methionine Residue ◽

Isoelectric Points ◽

Cytosolic Glutathione ◽

Lung And Kidney

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.

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A novel glutathione transferase (13–13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes

Biochemical Journal ◽

10.1042/bj2780137 ◽

1991 ◽

Vol 278 (1) ◽

pp. 137-141 ◽

Cited By ~ 70

Author(s):

J M Harris ◽

D J Meyer ◽

B Coles ◽

B Ketterer

Keyword(s):

Rat Liver ◽

Glutathione Transferase ◽

Ethacrynic Acid ◽

Gel Filtration ◽

Cumene Hydroperoxide ◽

Structural Similarity ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Linoleic Acid Hydroperoxide ◽

The Matrix

A rat liver mitochondrial-matrix fraction was prepared and shown to have 1-chloro-2,4-dinitrobenzene(CDNB)-metabolizing glutathione transferase (GST) activity. Further fractionation by sequential gel filtration, isoelectric focusing or chromatofocusing and hydroxyapatite chromatography yielded three GSTs of pI 9.3, 8.9 and 7.5, none of which bound to a GSH-agarose affinity matrix. Most of the activity was associated with the pI-9.3 form, which was selected for further study. Its activity was tested with the following potential substrates in addition to CDNB: 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, trans-4-phenylbut-3-en-2-one, 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, menaphthyl sulphate, cumene hydroperoxide, linoleic acid hydroperoxide and 4-hydroxynon-2-enal. Appreciable activity was obtained only with CDNB and ethacrynic acid (82 and 26 mumol/min per mg of protein respectively). The apparent Km for GSH, using 1 mM-CDNB, was 1.9 mM. The enzyme is a dimer of subunit Mr 26,500. It has a free N-terminus, which has enabled the first 33 amino acids to be sequenced. This portion of primary structure has a sequence in common with members of the Theta class of GSTs (eg. 36% identity with subunit 12) and also a sequence which might function as a mitochondrial import signal. It is novel and has been named ‘GST 13-13’.

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Kinetic independence of the subunits of cytosolic glutathione transferase from the rat

Biochemical Journal ◽

10.1042/bj2310263 ◽

1985 ◽

Vol 231 (2) ◽

pp. 263-267 ◽

Cited By ~ 89

Author(s):

U H Danielson ◽

B Mannervik

Keyword(s):

Glutathione Transferase ◽

Ethacrynic Acid ◽

Glutathione Transferases ◽

Kinetic Properties ◽

Subunit Interactions ◽

Steady State Kinetics ◽

Binding Isotherms ◽

Cytosolic Glutathione ◽

Kinetics Of ◽

Rate Behaviour

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

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CYTOSOLIC RAT LIVER GLUTATHIONE TRANSFERASE 4-4. Primary structure of the protein reveals extensive differences between homologous glutathione transferases of classes Alpha and Mu

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1986.tb09589.x ◽

1986 ◽

Vol 156 (2) ◽

pp. 349-350

Author(s):

Per Alin ◽

Bengt Mannervik ◽

Hans Jornvall

Keyword(s):

Rat Liver ◽

Primary Structure ◽

Glutathione Transferase ◽

Glutathione Transferases ◽

Liver Glutathione

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Characterization of Affinity-Purified Isoforms ofAcinetobacter calcoaceticusY1 Glutathione Transferases

The Scientific World JOURNAL ◽

10.1155/2014/750317 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Chin-Soon Chee ◽

Irene Kit-Ping Tan ◽

Zazali Alias

Keyword(s):

Hydrogen Peroxide ◽

Molecular Weight ◽

Gel Electrophoresis ◽

Ethacrynic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Acinetobacter Calcoaceticus ◽

Glutathione Transferases ◽

Glutathione S Transferase ◽

Substrate Specificities

Glutathione transferases (GST) were purified from locally isolated bacteria,Acinetobacter calcoaceticusY1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, andtrans,trans-hepta-2,4-dienalwhile GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) ofAcinetobacter calcoaceticusstrain PHEA-2, respectively.

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