• Title/Summary/Keyword: Trypsin

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Expression and Role of Trypsin-Like Enzyme Involved in Hatching of Preimplantation Mouse Embryos (생쥐 배아의 부화에 관여하는 Trypsin 유사 효소의 발현과 역할)

  • Kim, Soo-Kyung;Kang, Hee-Kyoo;Jun, Jin-Hyun;Choi, Kyoo-Wan;Kim, Moon-Kyoo
    • Development and Reproduction
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    • v.5 no.1
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    • pp.17-21
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    • 2001
  • This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

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Production and Purification of Trypsin Inhibitor from Streptomyces S-217 (Streptomyces S-217에 의한 Trypsin 저해물질의 생산 및 정제)

  • 류병호;이주화;신동분;김동석
    • Microbiology and Biotechnology Letters
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    • v.20 no.5
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    • pp.534-542
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    • 1992
  • Trypsin inhibtor produced by Streptomyces sp. S-217 was purified by solvent extraction and various column chromatographies. and physico-chemical properties of the inhibitor were investigated. Inhibitor complex was formed for incubation of 10 min. Streptomyces 5-217 showed the highest production of trypsin inhibitor when it was cultivated at $37^{\circ}C$ for 66 hr in the medium containing 2% mannitol & 0.9% peptone, pH 7.0. Trypsin inhibitor was purified by column chromatography and high performance liquid chromatography. Trypsin inhibitor indicated the maxium wavelength at 215 nm and solubilities in water, methanol and dimethyl sulfoxide were 95, 70 and 75%, respectively. The concentration of 50% inhibition ($IC^{50}$) was 15 $\mu$g/ml. The inhibitor was stable on heating at $100^{\circ}C$ for 60 min in pH 5~9 and was more stable in alkaline region than acidic region.

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Proteolytic Properties of Cathepsin L, Chymotrypsin, and Trypsin from the Muscle and Viscera of Anchovy, Engraulis japonica (멸치 육과 내장으로부터 분리한 Cathepsin L, Chymotrypsin 및 Trypsin의 단백질분해 특성)

  • PYEUN Jae-Hyeung;HEU Min-Soo;CHO Deuk-Moon;KIM Hyeung-Rak
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.28 no.5
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    • pp.557-568
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    • 1995
  • Proteolytic properties of enzymes from the muscle and viscera of anchovy have been examined. Cathepsin L, chymotrypsin, and trypsin showed similar Km values for casein. However, they had higher Km values for myofibrillar proteins than those for casein. The $k_cat$ of cathepsin L and chymotrypsin for myofibrillar proteins were higher than that of trypsin, and also cathepsin L and chymotrypsin caused higher hydrolysis in myofibrillar proteins of anchovy and yellowtail. In the presence of sodium chloride$(0-25\%)$, proteolytic activity for myofibrillar proteins from yellowtail was higher than that for casein. Proteolytic activity was decreased with the increase of sodium chloride concentration. Cathepsin L had been less affected by NaCl concentration and temperature on the hydrolysis of myofibrillar proteins than chymotrypsin and trypsin. Cathepsin L and chymotrypsin were move responsible to the autolysis of muscle proteins from fish than trypsin.

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Purification and Characterization of Trypsin Inhibitor from Alismatis Rhizoma (택사(Alismatis Rhizoma) trypsin inhibitor의 정제와 특성)

  • 박종옥;이인섭
    • Journal of Life Science
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    • v.12 no.2
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    • pp.151-157
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    • 2002
  • A trypsin inhibitor was isolated and purified from Azismatis Rhizoma which has been used as a galenic for diuretic and antiphlogistic. Purification was carried out by 0-80% saturated ammonium sulfate salting out, DEAE- cellulose ion exchange chromatogrphy, Sephadex G-150 gel filtration. The molecular weight of Alismatis Rhizoma trypsin inhibitor(ARTI) was estimated to be about 23,000 Da by gel filtration and SDS-PAGE, it must be monomer. ARTI was stable at 0~6$0^{\circ}C$, but at higher temperature its activity was decreased about 35%. When benzoyl-dl-arginine p-nitroanilide was used as a substrate of trypsin, half-maximal inhibition of ARTI was observed at 0.071 $\mu$M. ARTI inhibited the hydrolysis of trypsin non-competitively and Km value was 0.81 $\mu$M.

Preparation of Monoclonal Antibodies for Canine Trypsin-Like Immunoreactivity (개 트립신양(樣) 면역반응성 단클론 항체의 제작)

  • Kim, Sung-Soo;Kang, Ji-Houn;Cheong, Kwang-Myun;Yoo, Jai-Cheol;Chong, Chom-Kyu;Yang, Mhan-Pyo
    • Journal of Veterinary Clinics
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    • v.25 no.5
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    • pp.317-323
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    • 2008
  • Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.

Purification and Characterization of Trypsin Inhibitor from Alismatis Rhizoma and its Binding Protein, 10-Formyltetrahydrofolate Dehydrogenase (택사(Alismatis Rhizoma)로부터 트립신 저해제의 정제와 특성 규명 및 이와 결합하는 단백질, 10-Formyltetrahydrofolate Dehydrogenase에 관한 연구)

  • Kim, Ji-Man;Park, Jong-Ok;Shin, Young-Hee
    • YAKHAK HOEJI
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    • v.52 no.1
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    • pp.79-84
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    • 2008
  • Alismatis Rhizoma has been used as diuretics and antiphlogistics in the Chinese oriental medicine. A trypsin inhibitor was isolated from Alismatis Rhizoma using DEAE ion exchange column, trypsin affinity column, and FPLC chromatography, and its activity and characteristics were studied. The purifed Alismatis Rhizoma trypsin inhibitor (ARTI) was estimated to be about 22 kDa. The sequence determination on N-terminal amino acid residues and 84 amino acid residues has been completed, yet no homology has been found with trypsin inhibitors reported at NCBI. ARTI did not show inhibitory activities on chymotrypsin and elastase, however it exhibited a significant inhibitory activity on bovine trypsin, and formed a complex with rat liver 10-formyltetrahydrofolate dehydrogenase.

Effects of Trypsin Inhibitors on Oleic acid Induced Acute Pancreatitis in Dogs (개에서 Oleic acid로 유발시킨 급성췌장염에 대한 Trypsin inhibitor의 투여효과)

  • 윤영민;최희인;조명행
    • Biomolecules & Therapeutics
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    • v.5 no.2
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    • pp.158-164
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    • 1997
  • To investigate the effects of trypsin inhibitors, aprotinin and urinary trypsin inhibitor (UTI), on the cute pancreatitis, this study was carried out in dogs of acute pancreatitis induced by oleic acid (0.28 mg/kg). Administration with aprotinin and UTI seemed to have a therapeutic effect on the clinical sign, ultrasonographic finding, histopathologic finding. But in amylase and lipase activity, there were no significant differences among three groups.

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Protein Analysis Using a Combination of an Online Monolithic Trypsin Immobilized Enzyme Reactor and Collisionally-Activated Dissociation/Electron Transfer Dissociation Dual Tandem Mass Spectrometry

  • Hwang, Hyo-Jin;Cho, Kun;Kim, Jin-Young;Kim, Young-Hwan;Oh, Han-Bin
    • Bulletin of the Korean Chemical Society
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    • v.33 no.10
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    • pp.3233-3240
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    • 2012
  • We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

Citrus unshiu Water Extract Inhibits Trypsin-induced $TNF-{\alpha}$ and Tryptase Productions by Blocking the ERK Phosphorylation and Trypsin Activity

  • Kang, Ok-Hwa;Kim, Dae-Ki;Lee, Young-Mi
    • Natural Product Sciences
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    • v.10 no.5
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    • pp.211-216
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    • 2004
  • Citrus unshiu (Rutaceae) has long been known as an anti-inflammatory and anti-allergic agent. In the present study, the inhibitory effect of CUWE (Citus unshiu water extract) on the production of $TNF-{\alpha}$ and tryptase was examined. In addition, a possible mechanism for the inhibition of trypsin-stimulated human leukemic mast cell-1 (HMC- 1 ) activation was determined. To do so, $TNF-{\alpha}$ production from the HMC-1 cells that were stimulated by trypsin (100 nM) in the presence or absence of CUWE $(10,\;100,\;and\;100\;{\mu}g/ml)$ was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. The tryptase production was evaluated by reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. Results showed that the CUWE inhibited production of both $TNF-{\alpha}$ and tryptase from the trypsin-stimulated HMC-1 in a dose-dependent manner. The CUWE a1so inhibited the ERK phosphorylation and trysin activity. These results indicate that the CUWE had an inhibitory effect on $TNF-{\alpha}$ and the tryptase productions by blocking the ERK phosphorylation and trypsin activity.

Immobilization of Trypsin on Chitosan Nonwoven Using Glutaraldehyde (글루타알데하이드에 의한 키토산 부직포에 트립신 고정화)

  • Kim, Jung Soo;Lee, So Hee;Song, Wha Soon
    • Journal of the Korean Society of Clothing and Textiles
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    • v.37 no.7
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    • pp.852-863
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    • 2013
  • We investigate the immobilization of trypsin on chitosan nonwoven using glutaraldehyde (GA). The conditions for trypsin on chitosan nonwoven and GA cross-linking were optimized depending on different conditions. The order of GA cross-linking was determined by the activity of immobilized trypsin. The characteristics of chitosan nonwoven were examined by Fourier-transform infrared (FT-IR) and surface morphology analyses (SEM). Results showed that the optimal treatment conditions for trypsin on chitosan nonwoven were as follows: pH 8.5; temperature $37^{\circ}C$; trypsin concentration 15% (o.w.f); and treatment time 60 min. Those for GA cross-linking were: pH 10.0; GA concentration 3% (v/v); and treatment time 120 min. FT-IR analysis showed that GA was cross-linked on chitosan nonwoven. The SEM analysis also showed that trypsin was immobilized on chitosan nonwoven.