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Identification of Thioredoxin reductase in nucleus of HeLa cell

Identification of Thioredoxin reductase in nucleus of HeLa cell
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대학원 분자생명과학부
이화여자대학교 대학원
Thioredoxin reductases are homodimeric flavoproteins that catalyze the reduction of thioredoxin by NADPH. Three distinct forms of mammalian thioredoxin reductases are in cytoplasm, mitochondrium and microsome respectively. However, thioredoxin reductase in nucleus was not found yet, even though thioredoxin functions as a redox regulator in nucleus. In this thesis, the presence of thioredoxin reductase in nucleus was investigated. Thioredoxin reductase activity in nuclear extract was estimated to be 0.056 mmoles/ by spectrophotometric assay and the activity was confirmed by redox western blot assay. The nuclear thioredoxin reductase was isolated by 2'5' ADP-sepharose^(TM) 4B, phenyl sepharose, TSK-gel DEAE 5PW column chromatography. The enzyme was solubilized in the buffer containing either NaCl or Nonidet p-40. In contrast to the known thioredoxin reductases, the nuclear enzyme can bind 2'5' ADP-sepharose^(TM) 4B in the presence of ammonium sulphate. These properties suggest that the nuclear enzyme is a new type of thioredoxin reductase.;Homodimeric flavoprotein인 thioredoxin reductase는 NADPH로부터 전자를 받아thioredoxin을 환원시키는 enzyme이다. 3가지 형태의 mammalian thioredoxin reductase 는 cytoplasm과 mitochondrium 그리고 microsome에 각각 존재한다. Thioredoxin이 nucleus에서 redox regulator로 작용함에도 불구하고, nucleus에 존재하는 thioredoxin reductase는 아직 발견되지 않았다. 본 논문에서, thioredoxin reductase activity를 지니는 enzyme이 Hela cell 의 nucleus 에 존재하는지 조사하였다. Nuclear extract의 thioredoxin reductase activity 는 spectrophotometric assay와 redox western blot assay를 통해서 확인되었는데, spectrophotometric assay를 통해 측정된 activity 는 0.056 mmoles/ 이었다. Activity를 지니는 이 enzyme은 2'5' ADP-sepharose^(TM) 4B, phenyl sepharose, TSK-gel DEAE 5PW, phenyl superose HR5/5 column chromatography를 통해 분리되었다. 이 enzyme은 NaCl 이나 Nonidet p-40 가 포함된 buffer에 녹는 성질을 가지고 있으며 이미 알려진 thioredoxin reductases와는 다르게 ammonium sulfate가 포함되어 있는 상태에서도 2'5' ADP-sepharose^(TM) 4B에 붙을 수 있다. 이러한 성질은, nucleus에 존재하는 enzyme이 새로운 형태의 thioredoxin reductase일 가능성을 나타낸다.
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