• Applied Biological Chemistry
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• v.25 no.3
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• pp.182-188
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• 1982

A fluorometric method is described which permits the assay of trypsin inhibitor contained in a sample in an extremely small amount, utilizing a novel reagent generally called fluorescamine. The fluorometric assay with an enzyme kinetic approach has been found to be at least 100 times more sensitive than the well-known Kunitz's spectrophotometric method, considerably taster and less complicated, when it was demonstrated with the anti tryptic activities of very dilute extracts from soybean, red-bean and mung bean. Details of experimental procedure as well as theoretical considerations will be discussed.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1977.10a
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• pp.195.4-196
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• 1977

Trypsin에 대해서 비해물질을 생성하는 Streptomyces속 균주 AS 707을 선별하고 이 균주가 생성하는 물질의 작용성 및 제성질을 조사한 결과 본 저해물질은 열에 대해서 비교적 강하고 pH 처리에 대해서는 산성에서 중성에 걸쳐서는 안정하나 alkali성에서는 불안정했고 $\alpha-chymotrypsin과$ papain에 대해서도 저해작용을 하였으며 trypsin에 대한 저해양상은 mixed type이었다.(중략)

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2079-2094
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• 2018

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.23 no.1
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• pp.12-24
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• 1990

Two trypsin-like enzymes, designated trypsin A and 3, purified from the intestine of menhaden by $(NH_4)_2SO_4$ fractionation, Benzamidine-Sepharose 6B affinity chromatography, DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The two trypsins were subjected to compare the enzymatic properties of the trypsin-like enzymes from the other dark fleshed fishes. Both trypsins catalysed the hydrolysis of N$\alpha$-benzoyl-DL-arginine-p-nitroanilide and they were remarkably inhibited by several well known trypsin-inhibitors, tosyllysyl chloromethyl ketone, soybean trypsin inhibitor, be-nzamidine, leupeptin and antipain, etc. Therefore, it was ascertained that the two enzymes are serine-type trypsins. The molecular weights of these enzymes were about 25,000 and 26,200, respectively, ;Is determined by SDS-PAG electrophoresis and by Sephadex G-100 gel filtration, and the molecular weights of these two enzymes are somewhat fewer than those from the other dark fleshed fishes. Both enzymes had less basic amino acids such as arginine and Iysine, whereas they had slightly high contents of neutral amino acids, glycine, alanine and tryptophane. The enzymes showed a pH optimum of $8\~11$ at $60^{\circ}C$ against the $N\alpha$-benzoyl-DL-argi-nine-p-nitroanilide substrate and they were quite unstable above $40^{\circ}C$ and under the atidic pH region. The Km constant of the two enzymes against the $N\alpha$-benzoyl-DL-arginine-p-nitroanilide was $1.4\times10^{-4}M$ for trypsin A and $4.3\times10^{-5}M$ for trypsin B, respectively.

• Applied Biological Chemistry
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• v.20 no.2
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• pp.182-187
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• 1977

This investigation was to determine the changes in the trypsin inhibitor activity(TIA) and electrophoresis patterns of the soybean cotyledon and axis during germination. The TIA of the cotyledon decreased slightly and that of the axis decreased rapidly to 50% activivity after 7 day germination. At the 2nd, 3rd and 4th day's germination the TIA of the defatted dry axis was higher than that of cotyledon. However, the TIA of the fresh cotyledon was lower than that of the axis, due to its higher moisture content. Results from the electrophoretic studies showed that band 1 (polymer, 15S etc.), 2(11S), and 3(7S) whichare the major reserve proteins of soybean were decreased consid erably in cotyledon and axis and the fragments with Rm values between 0.5 and 1.0 were increased and band 5 showed up during germination. The band 4 of the cotyledon and band 6 of axis were not changed during germination. Generally speaking, the TIA and thereserve protein decreased as germination proceed.

• Journal of Pharmaceutical Investigation
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• v.30 no.3
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• pp.159-166
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• 2000

Kunitz-type soybean trypsin inhibitor (SBTI) and chondroitin sulfate (A, and C type) were conjugated using sodium periodate method. And the physicochemical, pharmacokinetic properties and immunogenecity of the conjugates (Chon-A-SBTI or Chon-C-SBTI) were characterized. We expected the conjugation using chondroitin sulfate to reduce the immunogenecity and to improve the pharmacological effect. As the results, the mean molecular weight of the conjugate highly increased. After I.V. injection of the radiolabeled conjugates or native SBTI into mice, it was found that native SBTI showed rapid elimination from plasma, whereas Chon-A-SBTI and Chon-C-SBTI were slowly eliminated. Organ distribution of the two agents at 30 min after I.V. injection was different : Chon-A-SBTI or Chon-C-SBTI accumulated to a large extent in the liver (13% in Chon-A-SBTI and 16% in Chon-C-SBTI), whereas native SBTI was taken up more rapidly by the kidney (107% dose/g of tissue) and excreated into the urine (26%). In addition we evaluated the therapeutic value of the conjugates by using the sublethal septic shock model caused by pseudomonal elastase and tested the immunogenecity by passive cutaneous anaphylaxis shock (PCA). The conjugates were more effective than native SBTI against pseudomonal elastase induced septic shock in guinea pig. In case of the conjugates, the pharmacological and therapeutic effect lasted over 3 hours long. In immunogenecity test, both of the conjugates showed the reduction of their immunogenecity, especially Chon-A-SBTI looked most effective.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.6
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• pp.1010-1015
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• 1995

Extent of reduction in protein digestibility by Godulbaegi(lxeris sonchifolia H.) kimchi powder appeared to be related to the kimchi weight-to-protein weight ratio, the kind of protein sources, part of plants and fementation period. In vitro digestibilities of protein were significantly(p<0.05) reduced as the freeze dried kimchi powder weight-to-protein ration increased from 0.5 : 1.0 to 1.0 : 1.0 for all Godulbaegi samples and protein sources. Overall digestibility of protein sources in the presence of kimchi powder, the reduction ranked in the following order : soybean>casein>beef>squid. Lower(p<0.005) reduction occurred for each protein source when raw plant was exchanged for kimchi products. Some greater reduction of digestibility was noted in young plants and leaf samples than ripe or root samples. Trypsin inhibitor, which expressed as soybena trypsin inhibitor, was inversely related(r=0.8437) to in vitro protein digestibility of casein in the presence of Godulbaegi kimchi powder. More than three times of total polyphenols contained in leaves than in roots. Young leaves had 30% more total polyphenols(37.64mg/g sample) than that in ripe ones. Soaking in 5% NaCl solution for 24 hrs was markedly reduced in total polyphenols as 73% for leaves and 33% for roots. Remarkable reduction in total polyphenols was not checked during fermentation followed after soaking. Trypsin inhibitor content correlated well(r=0.8873) with total polyphenols in all of Godulbaegi samples.

• Korean Journal of Food Science and Technology
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• v.12 no.1
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• pp.24-33
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• 1980

Trypsin inhibitor and hemagglutinating activities of some minor beans produced in Korea were determined in comparison with those of soybean and the effects of heat treatment on the activities were studied. The results are summarized as follows 1. The trypsin inhibitor activity (% [TU] inhibited/mg) of soybean, red bean, kidney bean, and mung bean were 79.9, 46.4, 43.2 and 17.7, respectively, on the dry weight basis and were 194, 222, 170 and 75, respectively, on the protein basis. 2. Heat destruction by boiling or autoclaving of trypsin inhibitor activity of red bean, mung bean, kidney bean, and soybean were $85{\sim}87,\;87{\sim}94,\;76{\sim}79\;and\;67{\sim}72\;%$, respectively. No significant difference was, however, observed in the effect between the two heating methods. 3. The hemagglutinating activity (unit/g) of kidney bean, soybean, mung bean and red bean were 48,300, 18,000, 136 and non-detectable, respectively, on the dry weight basis and were 190,600, 43,700, 581 and non-detectable, respectively, on the protein basis. Heat treatment destructed the hemagglutinating activity in all three beans.

• Tuberculosis and Respiratory Diseases
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• v.43 no.3
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• pp.359-366
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• 1996

Background : Eosinophilic leukocytes are prominent cellular participants in the pathogenesis of allergic disease and asthma. Chemotaxis is still a very useful method in evaluating the response of human eosinophil to novel modulators. Degranulated mast cells and activated T lymphocytes are responsible for the pathophysiology of asthma and tryptase is one of most important proteases released after activation of mast cells. The purpose of this study was to investigate the actions of trypsin and chymotrypsin on eosinophils in terms of chemotaxis and activation. Method : Eosinophils were isolated by negative immunoselection from the peripheral blood of atopic donors. Chemotaxis was studied by using micro-Boyden chambers and ECP release was assayed by fluoroimmunoassay. Results : Eosinophil showed a chemotactic response to trypsin. Maximal chemotactic response was with $1000{\mu}g/ml$ trypsin ($56.52{\pm}14.50$/HPF) which was comparable to PAP. But chymotrypsin showed no significant chemotactic response to eosinophils. Trypsin at the concentration of 10, 100, $1000{\mu}g/ml$ induced secretion of ECP, which at the concentration of $10{\mu}g/ml$ represented about 2.7 times of the spontaneous rate of release. Soybean protease inhibitor reduced trypsin induced ECP release. Conclusion : Trypsin can induce chemotactic response to eosinophils and activation of eosinophils that can induce secretion of ECP. On the contrary, chymotrypsin showed no direct effect on eosinophils. We propose a role of trypsin on the chemotaxis and activation of eosinophils.