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

The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.

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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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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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