• Applied Biological Chemistry
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• v.40 no.3
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• pp.196-201
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• 1997

Assuming that Asp225 is the substrate specificity determinant of Achromobacter pretense I (APl) which is lysine-specific serine protease, the 225th residue was substituted for other amino acids with a hope that the substrate specificity of a mutant API is altered. Furthermore, to maturate preform of mutant API autocatalytically, Lys(-1) was also replaced by Met, Asp, or Glu. However, all the mutants were not expressed, or accumulated as inactive precursor proteins. This result implicats that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase but the substrate specificity of API is not determined only by the nature of residue 225.

• Journal of Life Science
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• v.16 no.5
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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.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.299-306
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• 1996

The effects of antimetabolite 6-aminonicotinamide on levels of soluble proteins, free amino acids and protease activity in various tissues of quail have been in vestigated. The levels of soluble proteins m liver, heart and pectoral muscle were markedly lowered and the specific activity of protease in kidney and pectoral muscle was markedly increased. The concentrations of aspartic acid / asparagine, alanine, valine, methionine, isoleucine, leucine and lysine in the liver were markedly increased. En the kidney the concentrations of aspartic acid I asparagine, arginine, threonine, alanine, proline and lysine were markedly increased but those of glutamic acid I glutamine were decreased. The concentrations of glutamic acid / glutamine and serine in the heart were reduced but those in glycine and methionine were increased. In the pectoral muscle the concentration of arginine was decreased but the concentration of alanine and threonine was increased. The overall results suggest that antimetabolite 6-aminonicotinamide may act to enhance concentrations of amino acids related to the generation of energy and to depress the biosynthesis of some specific amino acids.

• Fisheries and Aquatic Sciences
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• v.1 no.2
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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.