• Applied Biological Chemistry
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• 제40권3호
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• pp.196-201
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• 1997

Lysine 특이적 serine protease인 Achromobacter protease I(API)의 기질특이성을 결정하는 아미노산 잔기가 255위치에 존재하는 가정하에 이 잔가에 변이를 도입하여 기질특이성의 변화 유무를 조사하였다. 그리고 pro-API이 자기촉매적으로 소화될 수 있도록 Lys(-1) 또는 다른 아미노산 잔기로 치환하였다. 그러나 예상과는 달리 제작된 모든 변이체는 발현되지 않았거나 불활성전구체로서 발현되었다. 이 결과로부터 225위치의 잔기는 API의 maturation과정에 있어 Iysylendopeptidase활성에 중요한 역할을 담당하고 있으나 APl의 기질특이성은 225위치 잔기의 특성에 한정되지 않을 가능성이 시사되었다.

• 생명과학회지
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• 제16권5호
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• 한국동물학회지
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• 제39권3호
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• pp.299-306
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• 1996

항 대사물질 6-aminonicotinamide가 메추리 조직의 수용성 단백질, 유리 아미노산 및 단백질 가수분해효소에 미치는 영향을 조사하였다. 간(P<0.05), 심장(P<0.01), 흉부근육 조직(P<0.05)의 수용성 단백질의 함량은 대조군에 비해 현저히 감소하였다. 신장과 흉부근육 조직의 단백질 가수분해효소의 비활성도는 대조군에 비하여 현저히 감소하였다. 신장과 흉부근육 조직의 단백질 가수분해효소의 비활성도는 대조군에 비해 현저히 증가하였다. (P<0.05). 간(P<0.05)의 aspartic acid / asparagine, alanine, valine, methionine, isoleucine, leucine, lysine의 농도는 대조군에 비해 증가하였다. 신장(P<0.05)의 acid / asparagine, alanine, threonine, alanine, proline과 lysine은 증가하였으나 glutamic acid / glutamine의 농도는 감소하였다. 심장에서는 glycine과 methionine 농도는 증가하였으나 glutamic acid / glutamine의 농도는 감소하였다. 흉부근육 조직에서는 arginine의 농도는 감소하였으나 (P<0.05) alanine과 threonine의 농도는 증가하였다 (P<0.05). 본 연구 결과는 항 대사물질인6-aminonicotinamide가 유리 아미노산의 농도를 증가시키므로써 기포 물질 대사에 필요한 에너지를 유지하는 것으로 사료된다.

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.209-215
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• 1998

Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.