• Title/Summary/Keyword: TLCK

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Characteristics of the Inhibitory Action of Protease Inhibitors on the Glucose-6-phosphate Transporter

  • Choi, Joon-Sig;Shin, Jeong-Sook;Choi, Hong-Sug;Park, Jong-Sang
    • BMB Reports
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    • v.30 no.2
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    • pp.157-161
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    • 1997
  • The present paper reports characteristics and specificity of the inhibitory action of $N^{\alpha}-tosyl-L-lysine-chloromethyl\;ketone$ (TLCK) and $N^{\alpha}-tosyl-L-phenylalanine-chloromethyl\;ketone$ (TPCK) on the glucose6-phosphate transporter of rat liver microsomes. The TLCK-induced inhibition was pH dependent. The inhibition constants for TPCK were determined by following pseudo-Lst order reaction mechanism. The inhibition was protected by preincubation with excess amount of glucose-6-phosphate. The results proved that (a) TLCK inactivates the microsomal glucose-6-phosphate transporter, (b) the inhibition results from the modification of sulfhydryl groups of the transporter.

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Physiological importance of trypsin-like protease during morphological differentiation of streptomycetes

  • Kim, In-Seop;Kang, Sung-Gyun;Lee, Kye-Joon
    • Journal of Microbiology
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    • v.33 no.4
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    • pp.315-321
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    • 1995
  • The relationship between morphological differentiation and production of trypsin-like protease (TLP_ in streptomycetes was studied. All the Streptomyces spp.In this study produced TLP just before the onset of aerial mycelium formation. Addition of TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP activity. Addition of 2% glucose to the Bennett agar medium repressed both the aerial mycelium formation and TLP production in S. abuvaviensis, S. coelicolor A3(2), S exfoliatus, S. microflavus, S. roseus, s. lavendulae, and S. rochei. However the addition of glucose did not affect S. limosus, S. felleus, S. griseus, S. phaechromogenes, and S. rimosus. The glucose repression on aerial mycelium formation and production of TLP was relieved by the addition of glucose anti-metabolite (methyl .alpha.-glucopyranoside). Therefore, it was concluded that TLP production is coordinately regulated with morphological differentiation and TLP activity is essential for morphological differentiation in streptomycetes. The proposed role of TLP is that TLP participates in the degradation of substrate mycelium protein for providing nutrient for aerial mycelial growth.

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The Effect of the Hydrogen Bond Network in the $S_1$-pocket on Catalytic Activity of Serine Protease, Achromobacter Protease I (API)

  • Lim, Seong-Il;Byun, Myung-Woo;Choi, Cheong
    • Journal of Microbiology and Biotechnology
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    • v.8 no.2
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    • pp.158-164
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    • 1998
  • Crystal structural analyses of the API-TLCK complex revealed that the ${\epsilon}$-amino group (NZ) of the lysyl part of TLCK forms hydrogen bonds with OD1 of $Asp^225$ which is a substrate specificity determinant of API, OG of $Ser^214$, O of $Ser^214$, OG1 of $Thr^189$, and O of $Thr^189$ l89/. The ${\beta}$-carboxyl oxygen of $Asp^225$ forms hydrogen bonds with the NE1 of $Trp^182$. From these observations, it is thought that besides $Asp^225$, $Thr^189$, $Ser^214$, and $Trp^182$ may also contribute to the steric specificity for lysine and high proteolytic activity of API. The side-chain hydroxyl groups of $Thr^189$ and $Ser^214$ were removed to elucidate the role of these hydrogen bonds in the $S_1$-pocket. The $k_{cat}$/$K_m$ of T189V, S214A, and T189V.S214A were decreased to 1/4, 1/3, and 1/46, respectively, of the value for native API. The decreased activities were mainly due to the increase of $K_m$. The CD and fluoroscence spectra of the three mutants were similar to those of wild-type API. With regards to the kinetic parameters ($K_i\;and\;k_2$) of mutants for the reaction involving TLCK and DFP, $k_2$decreased by increase of $K_1$ only. These results suggest that the decreased catalytic activity of these mutants is caused by the partial loss of the hydrogen bond network in the $S_1$-pocket. On the other hand, the similarity of enzymatic properties between W182F and the native enzyme suggests that the hydrogen bond between OD2 of $Asp^225$ and NE1 of $Trp^182$ is not directly related to the reaction of $Asp^225$ with the substrate.

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Kinetic Analyses for Enzymatic Properties of Trypsins Purified from Dark-Fleshed Fish (혈합육어 Trypsin의 효소적 성질에 대한 반응속도론적 해석)

  • CHO Deuk-Moon;HEU Min-Soo;KIM Hyeung-Rak;KIM Doo-Sang;PYEUN Jae-Hyeung
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.29 no.1
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    • pp.64-70
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    • 1996
  • Kinetic properties of typsins purified from dark-fleshed fish (anchovy, mackerel, yellowfin tuna, and albacore) were examined and analyzed on $benzoyl-_{D,L}-arginine-p-nitroanilide\;(BAPNA)$. The values of Km' and $k_{cat}$ of the purified trypsins from the four dark-fleshed fish were found to be $49.3{\mu}M$ and $90.9\;min^{-1}$ for anchovy, $53.7{\mu}M$ and $61.2min-^{-1}$ for mackerel A, $96.5{\mu}M$ and $76.6min^{-1}$ for mackerel B, $62.8{\mu}M$ and $46.6min^{-1}$ for yellowfin tuna, and $98.3{\mu}M$ and $47.7min^{-1}$ for albacore, respectively. The values of $K_i$ on $tosyl-_L-lysine$ chloromethyl ketone (TLCK) were determined to be $20.90{\mu}M$ for anchovy trypsin, $2.86{\mu}M$ for mackerel trypsin A, $3.90{\mu}M$ for mackerel trypsin B, $0.96{\mu}M$ for yellowfin tuna trypsin, and $1.82{\mu}M$ for albacore trypsin. Thus yellowfin tuna trypsin was the most sensitive to TLCK among all trypsins. The activities and catalytic efficiency of the trypsins purified from the temperate zone fish, anchovy and mackerel, were higher than those of the trypsins purified from yellowfin tuna and albacore which migrate widely from the tropic zone to the temperate zone.

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Trypsins from the Dark Fleshed Fish(Anchovy, Mackerel, Yellowfin Tuna and Albacore) 2. Enzymatic Properties and Thermal Stabilities (혈합육어(멸치, 고등어, 활다랭이 및 날개다랭이)의 Trypsin 2. 성질과 열 안정성)

  • 조득문;허민수;변재형
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.22 no.4
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    • pp.458-464
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    • 1993
  • In the present paper enzymatic properties of the trypsins from the four dark fleshed fish were compared with each other and thermal stabilities of the enzymes were also investigated. The trypsins from the dark fleshed fish showed their activity only in BA-p-NA substrate of the amide substrates such as BA-p-NA and SP-p-NA, and BAEE and TAME of the ester substrate such as ATEE, BAEE, BTEE, and TAME. The enzymes were strongly inhibited by the serine protease inhibitors such as antipain, leupeptin, TLCK, DFP and SBTI, and were also inhibited by such metal ions as Cu$^{2+}$ and Hg$^{2+}$, but fairly activated by $Mg^{2+}$. Denaturation constants of the enzymes were 13.4$\times$10$^{-4}$ sec$^{-1}$ for anchovy trypsin, 47.18$\times$10$^{-4}$ sec$^{-1}$ for mackerel trypsin A, 34.06$\times$10$^{-4}$ sec$^{-1}$ mackerel trypsin B, 42.28$\times$10$^{-4}$ sec$^{-1}$ for yellowfin tuna trypsin and 16.6$\times$10$^{-4}$ sec$^{-1}$ for albacore trypsin at 55$^{\circ}C$. The activation energies of the trypsins at a temperature range of 3$0^{\circ}C$ to 5$0^{\circ}C$ were estimated to be 13.91 ㎉/mole for anchovy trypsin, 11.61㎉/mo1e and 8.43㎉/mole for mackerel trypsin A and for mackerel typsin B, 4.35㎉/mole for yellowfin tuna trypsin, and 3.76㎉/mole for albacore trypsin.

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Induction and Inhibition of Amphibian(Rana dybowskii) Oocyte Maturation by Proteolytic Enzymes In vitro. (단백질분해효소들의 양서류 난자에 대한 성숙유도와 억제작용에 관하여)

  • 권혁방;고선근;박현정
    • The Korean Journal of Zoology
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    • v.33 no.1
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    • pp.53-62
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    • 1990
  • Fully grown amphibian oocytes undergo their maturation (germinal vesicle breakdown, GVBD) during in vitro follicle culture when they are stimulated with frog pituitary homogenate (FPH) or progesterone. Present experiments were designed to determine whether proteolytic enzymes are involved in the regulation of the matunation process. Treatment of a $\alpha$ -chymoiyypsin inhibitor, N a -tosyl-L-phenylalanine-chloromethyl-ketone(TP) to the oocytes exhibited a biphasic phenomenon, the induction of the maturation without added hormone at relatively low doses (0.001-1 $\mu$M) and inhibition of the hormone induced oocyte maturation at a high dose (100 $\mu$M). Treatment of a trypsin inhibitor, N a -tosyl-L-lysine-chloromethyl ketone(TLCK) to the oocytes did not induce the maturation, but rather suppressed the hormone induced oocyte maturation in a high dose(100 $\mu$ M). Treatment of exogenous iyypsin to the oocyte induced their maturation without added hormone in a dose dependent manner (0.001-1 $\mu$ M). The data presented here indicate that some proteolytic enzymes play a role in the regulation of the maturation(meiotic arrest or reinitiation) in amphibians.

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Purification and Characterization of Extracellular Proteinase Produced by Pseudomonas aeruginosa (Pseudomonas aeruginosa 세포질외 serine계열 단백질 분해효소의 정제 및 특성)

  • 이은실;송철용
    • Korean Journal of Microbiology
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    • v.29 no.6
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    • pp.345-352
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    • 1991
  • A serine proteinase of molecular weight 60 kd was purified from culture supernatant of P. aeruginosa using DEAE-Trisacryl M ion-exchange and AcA 54 gel filtration column chromatography, and the properties of serine proteinase were characterized. By means of SDS-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was 55 kd. The optimal pH for the activity of purified enzyme was 7.5. The activity of the purified enzyme was completely inhibited by Di-isopropylfluorophosphate(DFP) and N-.alpha.-p-tosyl-L-lysine choloromethyl detone(TLCK) but not by other proteinase inhibitors such as E-64, pepstatin A, 1, 10-phenanthroline. The purified enzyme was capable of degrading type I and type IV collagen. Antisera obtained from hymans infected with Pseudomonas aeruginosa reacted to the purified serine proteinase in immunoblots. These results indicate that the purified enzyme is trypsin-like serine proteinase and this enzyme of P. aeruginosa may play an important role in tissue damage as a spreading factor and may be useful for serodiagnosis of Pseudomonas infections.

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Characteristics of the Hemolytic Factor in the Body Fluid from Redworm, Lumbricus rubellus (지렁이 (Redworm*: Lumbricus ruellus) 체액내 용혈인자의 특성)

  • 손영종;이정우장정순
    • The Korean Journal of Zoology
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    • v.34 no.1
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    • pp.20-30
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    • 1991
  • 지렁이(L. rubelIus) 체액내의 적혈구용혈능깍 단백질분해능을 확인하였다. 지렁이의 체액 0. 33 $\mu$l속에 있는 용혈인자는 9 $\times$ 1011 rat RBCs를 2분만에 완전히 용혈시켰으며, 다른 포유류들의 적혈구에도 약간의 차이는 있으나 비슷한 용혈능을 보였고, 이 용혈인자는 지렁이의 혈구가 아닌 몸체조직에서 분비되는 것으로 생각되었다. 용혈인자는 pH 6.5-7.5사이에서 활성이 가장 강하였고, 63'c로 30분 열처리할 경우 활성이 완전히 없어졌으며, 2-mercaptoethanol은 용혈인자의 활성을 증가시켰다. 이 인자의 활성도는 여러가지의 당류, LPS, cholesterol, prosphatidyl Choline, Ch10ropromazine, Sphin90myelin 및 FG2+, Fe3+, CU2+, ZR2+ 등의 금속이온에 의하여 활성이 저해되었다. 한편 지렁이 체액내의 단백질 분해인자는 BSA와 19G를 여러 조각으로 분해시켰으며 이 분해인자는 PMSF및 TLCK에 의하여 활성이 억제되지 않았다. 지렁이의 체강에는 용혈소를 분비하는 박테리아들이 존재하였으나 이들 박테리아들의 용혈소는 지렁이의 용혈인자와는 전기영동이동도에서 차이가 있었다.

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Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

  • Kim, Se-Kwon;Park, Pyo-Jam;Kim, Jong-Bae;Shahidi, Fereidoon
    • BMB Reports
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    • v.35 no.2
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    • pp.165-171
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    • 2002
  • A serine collagenolytic protease was purified from the internal organs of filefish Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and $55^{\circ}C$. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

Fibrinolytic Enzyme Activity of Extract from Camellia japonica L. (동백나무 추출물의 혈전용해 효소활성)

  • Lim, Chae-Young;Lee, Sook-Young;Pyo, Byeong-Sik;Kim, Sun-Min
    • Korean Journal of Medicinal Crop Science
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    • v.14 no.4
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    • pp.195-201
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    • 2006
  • The fibrinolytic activities of soluble proteins extracted from young leaves of Camellia japonica L. were studied. Fibrinolvity activity of extract from partitions of C. japonica L. showed 1.6-2.0 times higher than plasmin used as positive control. The fibrinolytic enzyme was confirmed directly from young leaves of C. japonica L. by a fibrin Plate and fibrin zymography. The protein was composed of a single polypeptide and its apparent molecular weight was found to be 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the fibrinolytic activity were pH 5.5 and $30^{\circ}C$, respectively. Also, the fibrinolytic activity was clearly inhibited by PMSF and TLCK, suggesting that it is a member of the trypsin-like serine protease. All these results suggest the protease is a fibrinolytic enzyme belong to a family of trypsin-like serine protease.