|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,700,000 (Direct Cost: ¥1,700,000)
Thioredoxin and thioredoxin reductase were purified from bovine small intestine according to standard methods, including heat treatment, ammonium sulfate fractionation, acetic acid treatment at pH5.2, anion-exchange column chromatography, Sephacry S-100 gel-filtration, and CM-Sepharose column chromatography. Thioredoxin reductase was finally purified on affinity chromatography of bovine small intestine thioredoxin-ligand Sepharose 4B following ADP-Sepharose. Hydrogen peroxide caused cytotoxicity of the small intestine epitherial cell line, IEC-6, as judged from an MTT assay and the release of lactate dehydrogenase. Thioredoxin reductase activity and SH content decreased dose-dependently with H_2O_2, but thioredoxin activity increased at low H_2O_2 concentrations. In addition, the increase in thioredoxin activity was time-dependent during the initial stages of oxidative stress. Reverse transcription-polymerase chain reaction (RT-PCR) amplification also showed that the mRNA content in IEC-6 cells increased time-dependently at 0.25 mMH_2O_2. These results indicate that cellular oxidative shock causes an increase in the activity of thioredoxin, which is involved in the defense mechanism against oxidative stress.