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

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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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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Activation and inhibition of microsomal glutathione transferase from mouse liver

Biochemical Journal ◽

10.1042/bj2490819 ◽

1988 ◽

Vol 249 (3) ◽

pp. 819-823 ◽

Cited By ~ 30

Author(s):

C Andersson ◽

M Söderström ◽

B Mannervik

Keyword(s):

Molecular Mass ◽

Mouse Liver ◽

Glutathione Transferase ◽

Cross Reactivity ◽

Steep Slope ◽

Glutathione Transferases ◽

Inhibitor Concentration ◽

Cytosolic Glutathione ◽

Concentration Curves ◽

Microsomal Glutathione

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.

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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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Isoenzymes of glutathione transferase in rat kidney cytosol

Biochemical Journal ◽

10.1042/bj2300609 ◽

1985 ◽

Vol 230 (3) ◽

pp. 609-615 ◽

Cited By ~ 37

Author(s):

C Guthenberg ◽

H Jensson ◽

L Nyström ◽

E Österlund ◽

M K Tahir ◽

...

Keyword(s):

Rat Liver ◽

Liver Enzymes ◽

Glutathione Transferase ◽

Ethacrynic Acid ◽

Rat Kidney ◽

Glutathione Transferases ◽

Substrate Specificities ◽

Additional Peak ◽

Rat Kidney Cytosol ◽

Rat Liver 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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A preliminary characterization of the cytosolic glutathione transferase proteome from Drosophila melanogaster

Biochemical Journal ◽

10.1042/bj20111747 ◽

2012 ◽

Vol 442 (1) ◽

pp. 181-190 ◽

Cited By ~ 56

Author(s):

Chonticha Saisawang ◽

Jantana Wongsantichon ◽

Albert J. Ketterman

Keyword(s):

Drosophila Melanogaster ◽

Cell Line ◽

Glutathione Transferase ◽

Embryonic Cell ◽

Kinetic Properties ◽

Genome Database ◽

Reverse Transcription Pcr ◽

Unique Character ◽

Cytosolic Glutathione ◽

Processed Pseudogene

The cytosolic GST (glutathione transferase) superfamily has been annotated in the Drosophila melanogaster genome database. Of 36 genes, four undergo alternative splicing to yield a total of 41 GST proteins. In the present study, we have obtained the 41 transcripts encoding proteins by RT (reverse transcription)–PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all of the annotated DmGSTs (D. melanogaster GSTs) in the proteome are expressed in the late embryonic stages of D. melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8, in addition to two isoforms from the Delta class that have been published previously. Only approximately one-third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class.

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Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases

Biochemical Journal ◽

10.1042/bj2470707 ◽

1987 ◽

Vol 247 (3) ◽

pp. 707-713 ◽

Cited By ~ 117

Author(s):

U H Danielson ◽

H Esterbauer ◽

B Mannervik

Keyword(s):

Chain Length ◽

Active Site ◽

Glutathione Transferase ◽

Physiological Role ◽

Catalytic Efficiency ◽

Glutathione Transferases ◽

Side Chain ◽

Structure Activity ◽

Cytosolic Glutathione ◽

Free Energy Of Binding

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

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Glutathione transferases in rat hepatoma cells. Effects of ascites cells on the isoenzyme pattern in liver and induction of glutathione transferases in the tumour cells

Biochemical Journal ◽

10.1042/bj2570215 ◽

1989 ◽

Vol 257 (1) ◽

pp. 215-220 ◽

Cited By ~ 14

Author(s):

M K Tahir ◽

C Guthenberg ◽

B Mannervik

Keyword(s):

Glutathione Transferase ◽

Hepatoma Cell ◽

Normal Liver ◽

Hepatoma Cells ◽

Glutathione Transferases ◽

Trans Stilbene ◽

Cytosolic Glutathione ◽

Fold Induction ◽

Rat Hepatoma ◽

Rat Hepatoma Cells

Rat hepatoma cells grown intraperitoneally as an ascites tumour were analysed with respect to their contents of cytosolic glutathione transferases. In contrast with normal liver tissue, the hepatoma cells were dominated by the class Pi glutathione transferase 7-7. All the major hepatic enzyme forms were down-regulated to almost undetectable concentrations. Livers of rats bearing ascites-hepatoma cells expressed low, but significant, amounts of protein which, by electrophoretic and immunochemical properties, appeared identical with transferase 7-7. This enzyme is not detectable in normal hepatocytes. Treatment of rats with trans-stilbene oxide induced the expression of transferase 7-7 in the livers of normal rats as well as in hepatoma-cell-bearing animals. In addition, a 2-fold induction of transferase 7-7 was measured in the hepatoma ascites cells. No significant elevation of any other enzyme forms in the hepatoma cells was noted.

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Detoxification of Styrene Oxide by Human Liver Glutathione Transferase

Human Toxicology ◽

10.1177/096032718700600606 ◽

1987 ◽

Vol 6 (6) ◽

pp. 483-489 ◽

Cited By ~ 12

Author(s):

G.M. Pacifici ◽

M. Warholm ◽

C. Guthenberg ◽

B. Mannervik ◽

A. Rane

Keyword(s):

Isoelectric Focusing ◽

Glutathione Transferase ◽

Styrene Oxide ◽

High K ◽

Cytosolic Glutathione ◽

Human Livers ◽

Two Phases ◽

Kinetic Pattern ◽

Variable Substrate ◽

Kinetics Of

Cytosolic glutathione transferase (GST) was investigated in four human livers. The profile of GST activity was determined by isoelectric focusing using 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. Three livers contained at least one basic and a near-neutral isoenzyme (GST μ). GSTμ was not detectable in the fourth liver. The kinetics of GST with styrene oxide as the electrophilic substrate were studied in the cytosolic fraction, with the pooled fractions from isoelectric focusing containing high activity of GSTμ transferase, and with GSTμ purified to homogeneity. The cytosol obeyed Michaelis-Menten kinetics when styrene oxide was used as the variable substrate. The average (± s.e.m.) of the Vmax and Km were 21.9 ± 7.9 nmol min -1 mg-1 and 4.9 ± 0.4 mM, respectively. At varying concentrations of glutathione, the enzyme did not obey Michaelis-Menten kinetics. Such kinetics were also observed with the fractions from isoelectric focusing and with the homogeneous GSTμ fraction. The Eadie-Hofstee plot showed two phases: one with a low and another with a high Km value. The apparent K m values for the cytosol were 0.035 ± 0.022 and 0.88 ± 0.36 mM. The kinetic pattern of purified GSTμ is consistent with that found in the cytosol.

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