Purification of rat liver microsomal glutathione transferase

1991 ◽  
Vol 11 (3) ◽  
pp. 182-186
Author(s):  
Shi Nian ◽  
Liu Yu-gu
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Microsomal Glutathione

scholarly journals Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

1990 ◽  
Vol 272 (2) ◽  
pp. 479-484 ◽  
Author(s):  
C Andersson ◽  
R Morgenstern
Keyword(s):  
Chemical Modification ◽  
Rat Liver ◽  
Active Site ◽  
Glutathione Transferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Histidine Residues ◽  
Catalytic Function ◽  
Low Efficiency ◽  
Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

scholarly journals Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli

1995 ◽  
Vol 311 (3) ◽  
pp. 861-866 ◽  
Author(s):  
R Weinander ◽  
E Mosialou ◽  
J DeJong ◽  
C P D Tu ◽  
J Dypbukt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Expression Vector ◽  
Glutathione Transferase ◽  
Active Form ◽  
Coding Region ◽  
Western Blots ◽  
Cos Cells ◽  
Mammalian Expression ◽  
Microsomal Glutathione

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

scholarly journals Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

1991 ◽  
Vol 266 (4) ◽  
pp. 2076-2079
Author(s):  
C Andersson ◽  
E Mosialou ◽  
A E Adang ◽  
G J Mulder ◽  
A van der Gen ◽  
...  
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Microsomal Glutathione

Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation

1995 ◽  
Vol 1247 (2) ◽  
pp. 277-283 ◽  
Author(s):  
Claes Andersson ◽  
Fiorella Piemonte ◽  
Erifili Mosialou ◽  
Rolf Weinander ◽  
Tie-Hua Sun ◽  
...  
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Kinetic Studies ◽  
Microsomal Glutathione
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