Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes.
Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.
Wang
S T
ST
Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan 40203, Republic of China.
Chen
H W
HW
Sheen
L Y
LY
Lii
C K
CK
D000603:Amino Acids, Sulfur / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005721:Glutamate-Cysteine Ligase / Q000032:analysis / Q000378:metabolism; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000032:analysis* / Q000378:metabolism; D005980:Glutathione Reductase / Q000032:analysis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis* / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D008297:Male; D008715:Methionine / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000603:Amino Acids, Sulfur; D007527:Isoenzymes; D011506:Proteins; D008715:Methionine; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D005982:Glutathione Transferase; D002256:Carbonic Anhydrases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine