• Journal of Life Science
• /
• v.20 no.2
• /
• pp.183-189
• /
• 2010

A fibrinolytic enzyme producing Bacillus sp. J-19 was isolated from the popular Korean seasoning, pickled anchovy. The fibrinolytic enzyme was purified to homogeneity by chromatographic methods including ethanol precipitation and gel-filtration using Sephadex G-50. Compared to the crude enzyme extract, the specific activity of the enzyme increased 1021-fold with a recovery of 23%. The purified enzyme was estimated to be approximately 45 kDa by SDS-PAGE. Especially, the amidolytic activity in the presence of the synthetic substrate for serine protease (H-D-Ile-Pro-Arg-pNA, S-2288) represented approximately 17 U/mg. In addition, more than the 60% activity of the 45 kDa fibrinolytic activity was maintained in the presence of up to 30% (w/v) sodium chloride. These findings could provide a unique fibrinolytic enzyme, leading to a potential thrombolytic agent.

• Journal of Life Science
• /
• v.16 no.5
• /
• pp.716-722
• /
• 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.

• Proceedings of the Korean Biophysical Society Conference
• /
• 1998.06a
• /
• pp.23-23
• /
• 1998

To understand structural and functional basis of loaded energy in the metastable native structure of inhibitory serpins (serine protease inhibitors), we characterized mutations that decreased the loaded energy of ${\alpha}$$_1$-antitrypsin and simultaneously influenced its inhibitory activity. Various folding defects such as side-chain locking, buried polar groups in unfavorable hydrophobic environment, and cavities were found as the structural basis of the metastability of ${\alpha}$$_1$-antitrypsin in a region presumably directly involved in the formation of complex between the inhibitor and a target protease.(omitted)

• Proceedings of the Korean Biophysical Society Conference
• /
• 2001.06a
• /
• pp.31-31
• /
• 2001

The native strain of serpins (serine protease inhibitors) has been recognized as a mechanism of biological regulation. Indeed, some stabilizing single residue mutations of human $\alpha$$_1$-antitrypsin, a prototype serpin, relieved local strain and caused the loss of inhibitory activity. The native strain of $\alpha$$_1$-antitrypsin is distributed throughout the whole molecule, but the strain that regulates the function directly is highly localized in the regions that appear to be mobilized during complex formation with a target protease.(omitted)

• Proceedings of the Korean Biophysical Society Conference
• /
• 1997.07a
• /
• pp.40-40
• /
• 1997

$\alpha$$_1$- Antitrypsin is a key plasma serine protease inhibitor and its prime physiological role is an inhibitor of leukocyte elastase. The reactive center loop of $\alpha$$_1$-antitrypsin is characterized by the unusual mobility and the ability of insertion into the central $\beta$ sheet A. In particular the rate of loop insertion is considered to be critical for the formation of a stable complex between a serpin and its target protease.(omitted)

• Korean journal of food and cookery science
• /
• v.17 no.5
• /
• pp.497-502
• /
• 2001

능이버섯 〔Sarcodon aspratus(Berk.) S.Ito〕으로부터 단백질 가수분해 효소를 추출하여 75%(NH$_4$)$_2$SO$_4$ 염석과 DE52 anion exchange column chromatography 와 sepharyl-S 200 column 및 Mono s column chromatography 에 의해 정제하였는데 조 효소의 특이 활성은 55.2U/mg protein으로 조효소액에 비하여 11.26배 증가하였고 수율은 49.5%로 나타났다. 정제된 효소는 전기영동을 행한 결과 단일 band를 나타내었으며 분자량은 29,300으로 추정되었다. pH의 안정성은 4$^{\circ}C$에서 48시간 보존하였을 때 pH 8.5에서 가장 안정하였고, pH 5.5~10.5까지 높은 활성을 유지하였다. 온도에 대한 안정성은 30분간 보존 한 후 활성을 검토한 결과 단백분해능은 5$0^{\circ}C$까지 비교적 처음활성을 유지하다가 6$0^{\circ}C$에서는 53%정도의 활성이 유지되었으나 그 이상의 온도에서는 급격히 실화하여 7$0^{\circ}C$에서는 완전히 실화하였다. 금속 이온에 의해서는 크게 저해를 받지 않았으나 PMSF 저해제에 대하여 저해되어 본 효소가 serine protease임을 시사하였다.

• Proceedings of the Korean Society of Applied Entomolohy Conference
• /
• 2001.11a
• /
• pp.139-139
• /
• 2001

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.251-251
• /
• 2004

Plasminogen activators (PA)는 다수의 세포 형태에서 분비되는 것으로 알려진 serine protease이다. PA는 섬유소 용해, 배란, 유선 퇴화, 착상 및 수정 등 다양한 생리적인 과정에 관여한다. 본 연구는 난포액이 돼지 난자의 체외성숙에 미치는 영향을 검토하기 위하여, 다양한 조건하에서의 돼지 난자의 성숙과 난구세포-난자 복합체(Cumulus-Oocyte complexes: COCs) 또는 conditioned medium 내의 PA 활성을 검토하였다. 직경 2∼6m 난포로부터 COCs를 회수하여 일부는 난구세포를 제거하였다. (중략)

• Proceedings of the Korean Society of Sericultural Science Conference
• /
• 2001.10a
• /
• pp.67-67
• /
• 2001

no Abstract, See Full Text

• Proceedings of the Korean Society of Sericultural Science Conference
• /
• 2005.04a
• /
• pp.57-57
• /
• 2005