Transfection with gamma-glutamyl transpeptidase enhances recovery from glutathione depletion using extracellular glutathione.
Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is an important constituent of the antioxidant and detoxifying mechanisms of cells. The plasma membrane bound enzyme, gamma-glutamyl transpeptidase (GGT), catalyzes the first step in the degradation of extracellular glutathione, the components of which are then used for de novo glutathione synthesis. We tested the hypothesis that an increase in GGT activity would enhance the utilization of extracellular glutathione by cells challenged with a glutathione-depleting agent. A eukaryotic system stably overexpressing GGT (nearly 200-fold) was developed by transfection of NIH-3T3 fibroblasts with a human placental GGT cDNA. These cells and controls were incubated for 30 min with 1 mM diethyl maleate, which caused approximately 80% intracellular glutathione depletion. Glutathione was added to the medium and cells were allowed to resynthesize intracellular glutathione. The transfected cells used extracellular glutathione much more efficiently than controls in terms of both the concentration dependence and the rate of glutathione resynthesis. Serine-borate, a competitive inhibitor of GGT, blocked the restoration of intracellular glutathione. The results support the hypothesis that the increase in GGT activity that occurs in some toxicologic or pathologic conditions could provide protection against glutathione depletion.
Rajpert-De Meyts
E
E
Cell Biology Group of Childrens Hospital Los Angeles, California 90027.
Shi
M
M
Chang
M
M
Robison
T W
TW
Groffen
J
J
Heisterkamp
N
N
Forman
H J
HJ
D000818:Animals; D002460:Cell Line / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D004247:DNA / Q000494:pharmacology; D015894:Genome, Human; D005978:Glutathione / Q000096:biosynthesis* / Q000494:pharmacology; D006801:Humans; D051379:Mice / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis / Q000235:genetics*
D003470:Culture Media; D011994:Recombinant Proteins; D004247:DNA; D005723:gamma-Glutamyltransferase; D005978:Glutathione