• Title/Summary/Keyword: Trypsin

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Effects of Trypsin, Collagenase and Dimethyl Sulfoxide on Dissociation of Rat Heart Cells (배양을 위한 심근세포분리에 미치는 Trypsin, Collagenase와 Dimethyl Sulfoxide의 영향)

  • Park, Chang-Woo;Lee, Yung-Chang
    • Journal of Yeungnam Medical Science
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    • v.4 no.1
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    • pp.17-23
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    • 1987
  • New born rat heart cells were dissociated using trypsin and/or collegenase to elucidate the dissociation efficiency of these two enzymes. And the effect of dimethyl sulfoxide during and immediately after cell dissociation was also investigated to clarify the so-called protective activity of dimethyl sulfoxide on cell performance. The results can be summarized as follows. 1. Cold trypsin 18 hours pretreatment followed by warm collagenase treatment resulted best cell viability and cell yield. 2. Single, warm trypsin treatment gave the poorest result. 3. Dimethyl sulfoxide did not seem to play any protective role during or immediately after rat heart cell dissociation. It had very damaging effect on rat heart cells.

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RELATIONSHIP BETWEEN PROBING POCKET DEPTH AND TRYPSIN-LIKE ACTIVITY IN SUBGINGIVAL PLAQUE (치주낭 탐침깊이와 치은연하치태내 Trypsin-like activity와의 관계)

  • Park, Jea-Han;Han, Kyung-Yoon
    • Journal of Periodontal and Implant Science
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    • v.23 no.3
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    • pp.442-453
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    • 1993
  • In order to determine the relationship between probing pocket depth and trypsin-like activity in subgingival plaque, probing pocket depth and loss of attachment were measured by Michigan-O probe on mandibular incisors of 30 patients with adult periodontitis. And the trypsin-like activity of Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus was evaluated by the hydrolysis of N-Benzoyl-DL-Arginine-2-Naphthyla-mide (BANA) using PerioScan reagent cards(Oral-B Laboratories, Redwood City, CA). The obtained data were statistically analyzed by Microstat program. The results were as follows. 1. The number of teeth showing negative trypsin-like activity was more in shallow periodontal pocket groups, but the number of teeth showing positive trypsin-like activity was more in deep periodontal pocket groups. 2. There was a significant positive correlation between probing pocket depth and trypsin-like activity in subgingival plaque(y=0.413X - 0.955, r = 0.7024, p<0.001). 3. There was no consistent relationship between loss of attachment and trypsin-like activity in subgingival plaque(p>0.01).

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Inhibition of Trypsin-Induced Mast Cell Activation by Water Fraction of Lonicera japonica

  • Kang, Ok-Hwa;Choi, Yeon-A;Park, Hye-Jung;Lee, Joo-Young;Kim, Dae-Ki;Choi, Suck-Chei;Kim, Tae-Hyun;Nah, Yong-Ho;Yun, Ki-Jung;Choi, Suck-Jun;Kim, Young-Ho;Bae, Ki-Hwan;Lee, Young-Ml
    • Archives of Pharmacal Research
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    • v.27 no.11
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    • pp.1141-1146
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    • 2004
  • Lonicera japonica Thunb.(Caprifoliaceae) has long been known as an anti-inflammatory. In the present study, the effect of water fraction of Lonicera japonica (LJ) on trypsin-induced mast cell activation was examined. HMC-1 cells were stimulated with trypsin (100 nM) in the presence or absence of LJ (10, 100, and 1000 $\mu$ g/mL). TNF-$\alpha$ and tryptase production were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. LJ (10, 100, and 1000 $\mu$g/mL) inhibited TNF-$\alpha$ secretion in a dose-dependent manner. LJ (10, 100, and 1000 $\mu$g/mL) also inhibited TNF-$\alpha$ and tryptase mRNA expression in trypsin-stimulated HMC-1. Furthermore, LJ inhibited trypsin-induced ERK phosphorylation. However, LJ did not affect the trypsin activity even 1000 $\mu$g/mL. These results indicate that LJ may inhibit trypsin-induced mast cell activation through the inhibition of ERK phosphorylation than the inhibition of trypsin activity.

Effect of Red Ginseng Water Extract on Trypsin Activity (홍삼 물추출물이 Trypsin 활성에 미치는 영향)

  • Lee, Jong-Won;Kim, Na-Mi;Do, Jae-Ho
    • Journal of Ginseng Research
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    • v.28 no.3
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    • pp.127-131
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    • 2004
  • This study was carried out to investigate the effect of red ginseng water extract (RGWE) on trypsin activity. After extraction of fat soluble and saponin component from red ginseng powder by methyl alcohol, the residue was extracted with distilled water, and manufactured to water extract. The extract was dialyzed with different molecular cut off membrane. Trypsin activity demonstrated the highest level at the RGWE concentration of 9${\times}$10$\^$-2/% in reaction mixture, and also increased to 15% at 2.9${\times}$10$\^$-3/%. Km value was decreased and Vmax was increased in the present of red ginseng water extract. Red ginseng water extract was partially purified by dialysis, Bio-Gel P-I0 and DEAE-cellulose column chromatography. The active fraction demonstrated positive reaction to ninhydrin, DNS and folin reaction.

Characterization of Trypsin Inhibitors Purified from Trichosanthes kirilowii Root (하눌타리박의 뿌리로부터 분리 정제한 Trypsin Inhibitor들의 특성)

  • Park, Eun-Ju;Yun, Doo-Hee;Cho, Eun-Jyung;Ryu, Byung-Hho;Kim, Hee-Sook
    • Korean Journal of Food Science and Technology
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    • v.26 no.1
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    • pp.81-87
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    • 1994
  • Two different trypsin inhibitors, TRTI-1 and TRTI-2, were purified to near homogenity from Trichosanthes kirilowii root, by $0{\sim}90%$ saturated ammonium sulfate salting out, DEAE-Sephacel ion exchange chromatography, Sephadex G-50 gel filtration chromatography and trypsin-affinity chromatography. The molecular weight of TRTI-1 and TRTI-2 were estimated to be about 5,000 Da and 24,000 Da, respectively, by gel filtration and must be monomer and homodimer since they contain 4,000 Da and 10,000 Da each on SDS-polyacrylamide gel electrophoresis. TRTI-1 was stable after heating for at least 2 hr at $100^{\circ}C$ but TRTI-2 was completely inactivated after heating for 10 min at $90^{\circ}C$. When Bz-dl-Arg-pNA was used as a substrate of TPCK-treated trypsin, half-maximal inhibitions of TRTI-1 and TRTI-2 were observed at $0.8\;{\mu}M$ and 6\;${\mu}M$, repectively. Both TRTI-1 and TRTI-2 inhibited the hydrolysis of trypsin competitively and Km values were $0.97\;{\mu}M$ and $0.63\;{\mu}M$, respectively. Both TRTI-1 and TRTI-2 specifically inhibited trypsin but they did not inhibit other proteases tested, chymotrypsin, papain, elastase, collagenase, thermolysin, Nagarase, pepsin, and thrombin.

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Protease-activated Receptor 2 is Associated with Activation of Human Macrophage Cell Line THP-1

  • Kang, Chon-Sik;Tae, Jin;Lee, Young-Mi;Kim, Byeong-Soo;Moon, Woo-Sung;Kim, Dae-Ki
    • 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.

Antioxidant Effects of Noni (Morinda citrifolia) Extracts Treated with Hel and Trypsin (염산과 트립신으로 처리한 노니(Morinda citrifolia) 추출물의 항산화 효과)

  • Choi, Hye Young;Choi, Byung Chul;Sim, Sang Soo
    • YAKHAK HOEJI
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    • v.49 no.5
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    • pp.410-415
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    • 2005
  • To investigate biological activity of noni extracts treated with HCl and trypsin, we measured the antioxidant activity through vitro assay and cellular system. Both water and lipid soluble fraction of noni extracts dose-dependently scav­enged DPPH radical. Superoxide scavenging activity of lipid soluble fraction after treating HCl and trypsin was significantly more potent than those of other fractions in NBT/xanthine oxidase assay, which suggests that antioxidant activity of noni extracts was increased by the treatment with HCl and trypsin. In antioxidant assay using RBL 2H3 cells, water soluble frac­tion of noni extracts had little effect on silica-induced reactive oxygen species generation, whereas lipid soluble fraction inhibited in a dose dependent manner. In non-treated noni extracts, effect of water soluble fraction on silica/$CuSO_4$-induced lipid peroxidation was more potent than that of lipid soluble fraction. However, the effects of noni extracts were reversed in noni extracts treated with HCl and trypsin. These data suggest that water soluble substances may be converted into lipid soluble substances by the treatment with HCl and trypsin. From the above results, it is suggested that lipid soluble fraction of noni extracts contain antioxidant used in vitro assay and RBL 2H3 cellular system. Such an effect of noni extracts may be increased by the treatment with HCl and trypsin.

Comparative Biochemical Properties of Proteinases from the Hepatopancreas of Shrimp. -II. Purification of Trypsin from the Hepatopancreas of Penaeus orientalis-

  • Oh Eun-Sil;Kim Doo-Sang;Jung Kyoo-Jin;Pyeun Jae-Hyeung;Heu Min-Soo;Kim Hyeung-Rak
    • Fisheries and Aquatic Sciences
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    • v.1 no.2
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    • pp.209-215
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    • 1998
  • Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.

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Luteolin Inhibits Extracellular Signal-Regulated Kinase Pathway Through Protease-Activated Receptors (-2 and -4) and Their Agonist Activity

  • Lee, Sun-Hee;Sohn, Yong-Sun;Choi, Yeon-A;Lee, Ji-Eun;Kim, Dae-Ki;Lee, Young-Mi
    • Natural Product Sciences
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    • v.13 no.2
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    • pp.169-173
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    • 2007
  • Luteolin is a major flavonoid of Lonicera japonica and has anti-inflammatory effect. The activation of proteinase-activated receptor (PAR)-2 and -4 by trypsin appears to play a role in inflammation, In the present study, we examined the inhibitory effects of luteolin on activation of trypsin-induced human leukemic mast cells (HMC-1). HMC-1 cells were stimulated with trypsin, PAR-2 and PAR-4 agonist, in the presence or absence of luteolin. The level of TNF-${\alpha}$ secretion was measured by enzyme-linked immunosorbent assay (ELISA). The expression of tryptase and phosphorylated-extracellular signal-regulated kinase (ERK) were assessed by Westem blot analysis. Moreover, trypsin activity was measured by the substrate Bz-DL-Arg-p-nitroanilide (BAPNA). TNF-${\alpha}$ secretion and Tryptase expression in trypsin-stimulated HMC-1 cells were markedly inhibited by pretreatment of luteolin. Furthermore, the pretreatment of luteolin resulted in the reduction of ERK phosphorylation and trypsin activity. These results suggest that luteolin might has the inhibitory effects on the PAR-2 and -4-dependent inflammation.

Characterization of $\alpha$-amylase, Total Alkaline Pretense, Trypsin and Triacylglycerol-lipase Activity of the Euryhaline Rotifer Brachionus rotundiformis (해수산 rotifer, Brachionus rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin 및 triacylglycerol-lipase 활성 특성)

  • Kwon O-Nam;Park Heum-Gi
    • Journal of Aquaculture
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    • v.18 no.4
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    • pp.245-251
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    • 2005
  • This study was investigated the condition of their maximum activity to assay the enzymes of rotifer, Brachionus rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin and TG-lipase activities of rotifer were higher and more sensitive in phosphate-NaOH buffer than Tris-HCl buffer. $\alpha$-amylase, trypsin and TG-lipase activities were appeared the maximum at pH 8.0, and total alkaline protease activity showed the maximum activity at pH 7.0. $\alpha$-amylase activity showed the highest activity at $40^{\circ}C$, and total alkaline protease and trypsin activities were assayed the highest at $55{\~}60^{\circ}C$. However, TG-lipase activity was appeared the highest at $25{\~}30^{\circ}C$. The optimum substrate concentration of enzyme activity of a-amylase, total alkaline protease, rypsin and TG-lipase were $3.5\%$ starch, $\0.6%$ azo-casein, $87.5{\mu}M$ BApNA and 81.2 mM olive oil, respectively. The optimum reaction time of enzyme activity of $\alpha$-amylase, total alkaline protease, trypsin and TG-lipase were increased up to 40, 60, 30 and 25 min., respectively. The data obtained in this study could be used for the digestive enzyme research of rotifer, B. rotundiformis.