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백서 간장 세포 내 NADP-linked Isocitrate Dehydrogenase에 관한 연구

Other Titles
 Studies on the NADP - linked Isocitrate Dehydrogenase in the cytosol of rat liver cell 
 Dept. of Biochemistry and Molecular Biology (생화학-분자생물학교실) 
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저자는 전분 및 설탕식이가 간장세포에 존재하는 지방합성에 관여하는 효소로 알려진 isocitrate dehydrogenase(ICDH), glucose-6-phosphate dehydrogenase(G6PDH) 및 malic enzyme 활성에 어떻게 영향을 미치며 이 효소들 간에 상호관계를 규명하기 위하여 체중 200gm내외의 웅성 백서를 소정의 방법에 따라 정상식이(전분 62%, 지방 20%), 전분식이(전분 77%, 지방 5%), 설탕식이(설탕 77%, 지방5%)에 단백질, 비타민 및 mineral등을 일정량 첨가한 식이를 가지고 그 투여방법을 달리하여 사육한 백서 간장세포에서 G6PDH, malic enzyme, NADP-linked ICDH의 활성을 측정하였으며 백서 간장세포질에 존재하는 ICDH를 정제분리하여 다음과 같은 결론을 얻었다. 백서를 정상, 전분, 설탕식이군으로 나누어 각 군에 해당식이를 일주일간 계속 투여한 후 간장세포에서 G6PDH활성을 측정한 결과 효소활성은 정상식이군보다 전분식이군은 1.5배, 설탕식이군은 4배 증가하였다. Malic enzyme은

정상식이군이나 전분식이군에 비해 설탕식이군에서 효소활성이 2.5배 증가하였다.

그러나 ICDH 활성치는 별 차이가 없었으며 ICDH 활성치는 G6PDH나 malic enzyme 활성치보다 10배 높았다. 이런 조건에 있는 백서를 3일간 굶기고 정상, 전분, 설탕식이를 다시 각 해당 군에 3일간 재투여시 G6PDH 활성치는 정상식이군에 비해 전분식이군은 3배, 설탕

식이군은 4배 증가하였다. Malic enzyme 활성치는 정상식이군에 비해 전분 및 설탕식이군에서 효소활성이 1.4배 증가하였다. G6PDH나 malic enzyme은 설탕식이 및 백서를 굶긴 다음 식이를 재 투여시 효소활성이 현저하게 증가하였으나 ICDH 활성은 설탕식이 및 식이 투여방법에 의해 하등의 영향을 받지 아니한 사실로 미루어 보아 지방대사에 영향을 미치는 다른 lipogenic enzyme과는 달리 induce되는 효소가 아닌 constitutional enzyme으로 사료된다. 저자즌 Pantaloni(1973) 등이 소의 간장 세포에서 NADP-linked ICDH를 분리한 방법을 용이하고 간단하게 개량하여 백서 간장 세포에서 NADP-linked ICDH를480배 분리정제하였다.

이 저자의 방법은 Pantaloni(1973) 방법이나 Islam(1972) 방법보다 훨씬 간단하고 그정제율이 높았다.


Glucose-6-phosphate dehydrogenase(G6PDH), ATP_citrate lyase, malic enzyme and fatty acid synthetase have been shown as lipogenic and inducible enzymes by several investigators, however, NADP-linked isocitrate dehydrogenase(ICDH) in the cytosol is not well characterized in its biological, physical and molecular properties, but also not highly purified from the cytosol of rat liver cell yet.

Our experiments were designed to study whether ICDH is inducible enzyme by diets containing different composition of nutrients as does G6PDH, malic enzyme, and ATP-citrate lyase.

Finally our experiments were subjected for ICDH to purify from the cytosol of rat liver cell. The experimental results are summarized as follows.

Adult male rats(about 200gm of body weight) were divided into three groups; normal diet group(starch 62%, fat 20%), starch diet group(starch 77%, fat 5%) and sucrose diet group(sucrose 77%, fat 5%). In each diet, protein, vitamins and minerals were supplemented.

The specific activities of ICDH in the cytosol of liver cell of rats fed with normal, starch and sucrose diet for one week were 0.277 0.237 and 0.237 umoles/min/mg of protein, respectively. However, specific activities of G6PDH were increased 4-fold in sucrose diet group(0.10 umoles) and increased 1.5 fold in starch diet group(0.038 umoles) as compared to normal diet group(0.025 umoles). The specific activities of malic enzyme in the sucrose diet group(0.064 umoles) increased 2.5-fold as compared to normal diet group(0.025 umoles) and starch group(0.02 umoles).

The specific activities of ICDH in the cytosol of liver cell of rats after 3 days fasting followed by 3 days refeeding with respective diet were 0.270 umoles in normal group, 0.214 umoles in starch group and 0.213 umoles in sucrose diet group. These enzyme activities were similar to those of corresponding diet group of rats fed for one week continuously. However, specific activities of G6PDH were increased 3-fold in the starch diet group(0.218 umoles) and 4-fold in the sucrose diet group(0.273 umoles) as compared to control diet group(0.073 umoles).

The specific activities of malic enzyme in the starch and the sucrose diet group(0.064, 0.072 umoles, respectively) increased 1.4-fold as compared to the control diet group(0.046 umoles).

Our experimental results revealed that ICDH is not induced at all by starch and sucrose diet as did G6PDH and malic enzyme, indicating ICDH is to be a constitutional enzyme.

Furthermore, NADP-linked ICDH has been 480-fold purified from the cytosol of liver cell of rats by several steps of ammonium sulfate treatment, thermodenaturation, Sephadex G-150 gel filtration chromatography and DEAE-Sephadex A-50 chromatography.

Further experiments are in progress to characterize this enzyme.
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1. College of Medicine (의과대학) > Dept. of Biochemistry and Molecular Biology (생화학-분자생물학교실) > 2. Thesis
Yonsei Authors
Ahn, Yong Ho(안용호) ORCID logo https://orcid.org/0000-0002-4133-0757
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