• 한국수산과학회지
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• 제44권1호
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• pp.8-17
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• 2011

To identify and examine the distribution of proteolytic inhibitory activity in crude extracts from fish eggs, and to determine the applicability of these protease inhibitors as anti-degradation agents in surimi-based products and fish meat, we compared the inhibitory activities of various extracts from fish eggs to those of commercial proteases, such as trypsin and papain. We used the optimal conditions for the screening of trypsin activity: 30 ug/uL of 0.1% trypsin and 0.6 mM Na-benzoyl-L-arginine-p-nitroanilide (BAPNA) with a pH of 8.0 at $40^{\circ}C$ for 60 min. The activities of papain and four commercial proteases were investigated after mixing with 100 ug/uL enzymes and 0.3% casein with a pH of 8.0 at $40^{\circ}C$ for 60 min. We performed a screening assay to detect the inhibitory activity (%) of crude extracts from eight species of fish eggs against the target proteases trypsin and papain. The assay revealed a wide distribution of trypsin and papain inhibitors in fish eggs. The specific inhibitory activities (11.6.28.6 U/mg) of crude extracts from fish eggs against trypsin and BAPNA substrate were higher than that (0.64 U/mg) of egg whites, used as a commercial inhibitor. The inhibitory activities of crude extracts from fish eggs against trypsin, and of egg whites against casein substrate (1.94.4.51 U/mg), were higher than those of papain (0.24.1.57 U/mg) and commercial protease (0.04.0.32 U/mg). The extracts from fish eggs were rich in protease inhibitors that exhibited strong inhibitory activity against trypsin, a serine protease, and papain, a cysteine protease.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.59-67
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• 2023

Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A₂ in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A₂ activity. The ceramide synthase inhibitor Fumonisin B1, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A₂ inhibitor AACOCF₃ inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF₃, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A₂ in intestinal myofibroblasts.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1998년도 학술발표회
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• pp.23-23
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• 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)

• 분석과학
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• 제24권2호
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• pp.78-84
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• 2011

본 연구에서는 양성 및/또는 음성 이온 방식으로 저에너지 충돌-유발 분해(CID)를 이용한 serine protease 저해제인 camostat 와 그것의 분해산물인 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA)의 분해 패턴을 전자분무 소스가 있는 사중극자 텐덤 질량분석기(ESI-MS/MS)를 이용하여 최초로 조사하였다. Camostat의 양이온 CID 질량 스펙트럼 분석결과, 분자구조내 에스테르 결합을 이루는 카르보닐 기와 산소 원자사이의 단일 결합(C-O) 분해가 우선적으로 일어나고, guanidine 기의 초기 손실보다는 N,Ndimethylcarbamoylmethyl기의 초기 손실이 더 잘 일어난다는 것이 특징적으로 확인되었다. GBPA의 양이온 CID 스펙트럼의 경우는, 4-guanidinobenzoyloxy 기에 있는 카르보닐 기와 산소원자 사이의 초기 분해가 일어나서 m/z 145에서 가장 강도가 높은 피크를 만들었다. 반면에, GBPA의 음이온 스펙트럼은 m/z 312의 모분자 이온에서 $CO_2$와 NH=C=NH의 순차적인 중성 손실로 인하여 m/z 226의 가장 강도가 높은 피크가 특징적으로 생성되었다.

• Archives of Pharmacal Research
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• 제28권7호
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.15-22
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• 2009

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide(KCN)/0.1 mM iodoacetic acid(IAA), and membrane transport function and cell viability were evaluated by measuring $Na^+$-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in $Na^+$-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited $Na^+$-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a $Na^+$ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in $Na^+$-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers(dimethylthiourea and thiourea), and amino acids(glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase $A_2(cPLA_2)$. The ATP depletion-dependent arachidonic acid release was inhibited by $cPLA_2$ specific inhibitor $AACOCF_3$. ATP depletion-induced alterations in $Na^+$-dependent phosphate uptake and cell viability were prevented by $AACOCF_3$. Inhibition of $Na^+$-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and $cPLA_2$ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

• Molecular & Cellular Toxicology
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• 제2권3호
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• pp.212-215
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• 2006

Several reports have suggested a possible relationship between blood coagulation factors and schizophrenia. Plasminogen activator inhibitor type 1 (PAI-1) belongs to a serine protease inhibitor family, which regulates fibrinolysis and proteolysis by inhibiting plasminogen activation. The purpose of this study was to investigate the association of polymorphisms of the PAI-1 gene with schizophrenia in Korean population. Two important polymorphisms (-675 4G/5G and -844 G/A) located on promoter region of the PAI-1 gene were analyzed on 178 schizophrenia patients and 226 controls. The genotypic and allelic associations of -675 4G/5G were found significant. Furthermore, haplotype analysis revealed significant result, which suggests that -675 4G/5G polymorphism might confer increased susceptibility for schizophrenia in Korean population.

• Toxicological Research
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• 제17권
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.62-62
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• 2003

The native form of serpin (serine protease inhibitor) is kinetically trapped in metastable state. Metastability in these proteins is critical to their biological function. Serpins inhibit target proteases by forming a stable covalent complex in which the cleaved reactive site loop of the serpin is inserted into $\beta$-sheet A of the serpin with concomitant translocation of the protease to the opposite of the initial binding site. Despite recent determination of the crystal structures of a Michaelis protease-serpin complex as well as a stable covalent complex, details on the kinetic mechanism remain unsolved. In this study we constructed several $\alpha$$_1$-antitrypsin variants and examined their kinetic mechanism of loop translocation and formation of protease-serpin complex by stopped-flow experiments of fluorescence resonance energy transfer as well as quenched-flow experiment. We report here the relationship of serpin's conformational switch mechanism with Inhibitory activity. There is little direct correlation between loop insertion rate and inhibitory activity. Rather, disrupting a salt bridge between R196 and E354 accelerates loop translocation even though it impairs the inhibitory activity. Moreover, the serpin's reactive site loop is translocated, at least partially, prior to loop cleavage.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.