• Preventive Nutrition and Food Science
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• v.11 no.2
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• pp.127-132
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• 2006

Bacillus sp. strain K-l, which produces a strong fibrinolytic enzyme, was isolated from chongkukjang, a traditional Korean fermented soybean paste. The fibrinolytic enzyme was purified from chongkukjang base by using ammonium sulfate fractionation and chromatographic techniques. Purified enzyme, CK K-1 was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of a 12.4 kDa and a pI of 8.0. The optimal reaction pH value and temperature were 8.0 and $40^{\circ}C$, respectively. Phenyl-methyl-sulfonyl-fluoride (PMSF; serine protease inhibitor), ethylene-diamine-tetra-acetic acid (EDTA; metallo protease inhibitor), copper ion, ferric ion and lead ion inhibited the enzyme activity. These results indicated that the fibrinolytic enzyme is a metallo-serine protease and different from nattokinase and chongkukjangkinase.

• Journal of Life Science
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• v.16 no.4
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• pp.598-604
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• 2006

The $Ca^{2+}-activated$ neutral protease calpain induced proteolysis has been suggested to play a role in certain cell growth regulatory proteins. Cyclin proteolysis is essential for cell cycle progression. D-type cyclins, which form an assembly with cyclin-dependent kinases (cdk4 and cdk6), are synthesized earlier in G1 of the cell cycle and seem to be induced in response to external signals that promote entry into the cell cycle. Here we show that cyclin D3 protein levels are regulated at the posttranscriptional level by calpain protease. Treatment of human breast carcinoma MDA-MB-231 cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. The specific inhibitor of the 26S proteasome, lactacystin, the lysosome inhibitors, ammonium chloride and chloroquine, and the serine protease inhibitor, phenylmethylsulfonylfluoride (PMSF), did not block the degradation of cyclin D3 by lovastatin and actinomycin D. Results of in vitro degradation of cyclin D3 by purified calpain showed that cyclin D3 protein is degraded in a $Ca^{2+}-dependent$ manner, and the half-life of cyclin D3 protein was dramatically increased in LLnL treated cells. These data suggested that cyclin D3 protein is regulated by the $Ca^{2+}-activated$ protease calpain.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.1
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• pp.35-39
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• 1991

Properties of a protease purified from Neungee[Sarcodon aspratus(Berk, ) S. Ito] have been investigated. The enzyme displays a glycosylated serine protease. The enzyme is able to hydrolyze alanine glycine methionine glutamine and cysteine of N-CBZ and N-t-BOC-L-amino acid derivatibes relatively strongly but splits valine proline and isoleucine derivatives with low affinity which means the enzyme has the broad substrate spectrum toward the amino acids. Interestingly the enzyme was inhibited by bromelain inhibitor. That is the active site environ-ment of the enzyme is believed to be similar to that of bromelain However peptide mapping studies show that the two enzymes have distinct different cleavage sites.

• Proceeding of EDISON Challenge
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• 2016.03a
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• pp.86-89
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• 2016

The Zika virus, which is a member of the flavivirus genus, poses a serious threat to humanity because there is no vaccine or cure. Zika is suspected to cause microcephaly, and it is rapidly spreading throughout parts of Brazil. Surprisingly, there are no known protein structures for the virus which are essential for drug and vaccine development. This paper investigates the Zika virus's nonstructural proteins with template-based modeling by using GalaxyTBM/Refine/SC. GalaxyDock was used to examine the effectiveness of various known serine protease inhibitors in inhibiting the Zika viral protease. In testing five inhibitors, Kunitz soybean trypsin inhibitor showed the strongest binding affinity (-10.082 kcal/mol). This paper provides a rudimentary foundation for further drug discovery research regarding the Zika virus.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.3
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• pp.188-195
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• 2016

Neutrophils and lymphocytes are essential inflammatory cells in the pathogenesis of allergy. In this study, we evaluated the role of house dust mite (HDM) in the interaction between allergic lymphocytes and neutrophils. The extract of Dermatophagoides pteronissinus (DP) showed a stronger anti-apoptotic impact on neutrophil apoptosis in the coculture of allergic neutrophils with allergic lymphocytes when compared with that in allergic neutrophils alone. DP increased IL-6, IL-8, MCP-1, and GM-CSF in allergic lymphocytes, and the increased cytokines were inhibited by rottlerin-an inhibitor of the protein kinase C (PKC) ${\delta}$, as well as by SB202190-a p38 MAPK inhibitor. DP activated p38 MAPK in a time-dependent manner. The activation of p38 MAPK was suppressed by PAR2i, which is a protease-activated receptor (PAR) 2 inhibitor, and rottlerin. Both aprotinin-a serine protease inhibitor-and E64-a cysteine protease inhibitor-were not effective on cytokine secretion of lymphocytes. These results, despite increased cytokines in allergic lymphocytes via DP, did not show any differences between asthma and allergic rhinitis. Molecules, including cytokines, released by DP in lymphocytes inhibited the migration of neutrophils. This finding may further elucidate the pathogenic mechanism of allergic diseases due to HDM.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.4
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• pp.351-358
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• 2013

A protease inhibitor from fish eggs was fractionated using chromatographic methods. The fractionation efficiency was evaluated in terms of specific inhibitory activity (SIA, U/mg), purity (fold), total inhibitory activity (TIA, U), and recovery (%). The protease inhibitor (PI) from egg extracts of skipjack tuna (ST Katsuwonus pelamis), yellowfin tuna (YT Thunnus albacares) and Alaska pollock (AP Theragra chalcogramma) was fractionated using Sephadex G-50 gel filtration and DEAE-Sepharose CL-6B anion exchange chromatography based on protein size exclusion and net charge, respectively. Fractions exhibiting strong inhibitory activity were contained in the 30-50 kDa fraction on gel filtration and in the range of 0.4-0.7 M NaCl gradient fraction on anion exchange chromatography. The respective TIA and percent recovery of the fraction obtained with gel filtration toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 2,758.7 U and 29.6% for ST, 1,005.5 U and 25.6% for YT, and 1,267.5 U and 26.0% for AP. Gel filtration chromatography was more effective at fractionating PI than using ion exchange chromatography. These results suggest that fish eggs act as serine protease inhibitors and might be useful for protease inhibition in foodstuffs.

• IMMUNE NETWORK
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• v.5 no.4
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• pp.193-198
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• 2005

Background: Protease-activated receptor 2 (PAR2) belongs to a family of G protein coupled receptors activated by proteolytic cleavage. Trypsin-like serine proteases interact with PAR2 expressed by a variety of tissues and immune cells. The aim of our study was to investigate whether PAR2 stimulation can lead to the activation of human mac rophages. Methods: PAR2-mediated proliferation of human macrophage cell line THP-1 was measured with MTT assay. We also examined the extracellular regulated kinase (ERK) phosphorylation and cytokine production induced by trypsin and PAR2-agonist using western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Treatment of trypsin or PAR2-activating peptide increased cell proliferation in a dose-dependent manner, and induced the activation of ERK1/2 in THP-1 cells. In addition, trypsin-induced cell proliferation was inhibited by pretreatment of an ERK inhibitor (pD98059) or trypsin inhibitor (SBTI). Moreover, PAR2 activation by trypsin increased the secretion of TNF-${\alpha}$ in THP-1 cells. Conclusion: There results suggest that P AR2 activation by trypsin-like serine proteases can induce cell proliferation through the activation of ERK in human macrophage and that PAR2 may playa crucial role in the cell proliferation and cytokine secretion induced by trypsin-like serine proteases.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.165-170
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• 1995

The biochemical properties of the fibrinolytic protease of 50,800 Da isolated from the venom of Kgdistrodon blomhoffi brevicaudus were characterized. The enzyme hydrolyzed the carboxyl side of arginine in the synthetic chromogenic peptides, N-Benzoyl-Phe-Val-Arg-pNA and N-p-Tosyl-Gly-Pro-Arg-pNA, and the enzyme activity was inhibited by phenylmethylsulfonylfluoride indicating that the enzyme belongs to the serine protease family. The pretense showed maximum activity at pH 7.5 and inhibited by ZnCl$_2$, CuSO$_4$, but not by soybean trypsin inhibitor, pepstatin A, 2-mercaptoethanol and EDTA. The fm value determined with N-p-Tosyl-Gly-Pro-Arg-pNA was 0.2 mM. The thrombolytic activity of the purified enzyme was evaluated by platelet aggregation test in rabbits. While the platelet count ratio in blood of the rabbits injected with thrombin alone declined from 1.0 to 0.6 within 7 min and maintained around 0.6 for 24 hours thereafter, the ratio rapidly recovered from around 0.6 to 0.8 in 1 hr, to 1.0 in 24 hrs when the rabbits were sequentially treated with thrombin and the purified enzyme. The result showed that the serine protease from A. blomhoffi brevicoudus of 50,800 Da had a thrombolytic activity in vivo and the enzyme might be developed as a therapuetic agent for the treatment of thrombic disease.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.38-38
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• 1997

Serpins (serine protease inhibitor) are single polypeptide proteins of around 400 amino acids, and have a conserved secondary structure consisted of three ${\beta}$-sheets and nine ${\alpha}$-helices. Native conformation of inhibitory serpins is a metastable and requires conformational changes to inhibit target protease.(omitted)

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.