• Journal of Microbiology
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• v.33 no.4
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• pp.307-314
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• 1995

Trypsin like protease (TLP) and metalloprotease (MTP) were induced in associated with the mycelium differentiation in Streptomyces albidoflavus SMF301. TLP and MTP were purified and characterized from the culture. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The optimum pH and temperature of TLP were 10 and 40.$^{\circ}C$ Those of MTP were 8 and 55 $^{\circ}C$ TLP was stable at alkaline pH (6-9) and unstable above 45.$^{\circ}C$and MTP was stable at alkaline pH and unstable above 80.$^{\circ}C$ Km and Vmax values with benzoyl-arginyl p-nitroanilide of TLP were 139 $\mu$M, and 10 nmole of nitroanilide released per min per$\mu\textrm{g}$ protein, respectively. Km, and Vmax values with a synthetic substrate, leucine p-nitroanilide, or MTP were 58.9 $\mu$M, 3.47 nmol of nitroanilide released per min per$\mu\textrm{g}$protein, respectively. TLP was inhibited competitively by leupeptin; the inhibition constant was 0.0031 $\mu$M. MTP was inhibited by EDTA, phenonthroline and bestatin.

• Journal of Life Science
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• v.7 no.3
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• pp.228-233
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• 1997

A thiol protease has been isolated and partially purified from encysted brine shrimp Artemia franciscana using a four-step procedure(filtration, salting out, gel filtration and ion exchange chromatography). The optimum pH of the enzyme for caseinolytic activity was appeared to be 3.0, and the enzymematic activity was stable up to pH 6.0 but lost completely at the pH higher than 8.0. The optimal temperature of the enzyme was appeared to be 35$^{\circ}$C, and ninety percent of the enzyme activity was lost at 45$^{\circ}$C. Various metal ions, e.g., zinc, copper, iron, inhibited the enzyme activity; however, heavy metal chelator, e.g., EDTA, stimulated the enzyme activity. The protease was concluded to be a member of the thiol group protease, since it was inhibited by thiol protease inhibitors and iodoacetate. The protease was also concluded to be a acid protease based on optimum pH.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Journal of agricultural medicine and community health
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• v.22 no.1
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• pp.61-74
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• 1997

In case of tissue invading nematode, proteolytic enzyme was required at their parasitic life. Proteinases obtained from these parasites(Toxocara canis, Ansakis spp. and Trichinella spiralis) were extracted, isolated and further purified. And then the analysis for activity and inhibitory effect of proteinases were performed by appropriate substrate. Determination of protein as a circulating antigen was done in use of infected animal serum with above parasites, respectively. For above experimental objects, following procedures were performed. First, enzymatic activity was measured in use of azocasein and inhibitory effect of porteinase were studied by various inhibitors. Second, partially purified proteins containing enzymatic activity were obtained by ion exchange chromatography, ultrafiltration and electrophoretic elution. Third, role of the partially purified protein as a circulating antigen. The results obtained were as follows : 1. Enzymatic activity of each nematode proteinase was varied according to pH. Optimal pH of Toxocara canis, Ansakis spp. and Trichinella spiralis were pH 6.0, pH 5.5 and pH 6.5, respectively. The optimal molarity of buffer was 0.1M phosphate buffer. Although little difference between these proteinases was observed, temperature stability was at least maintained at $4^{\circ}C$ until 5 days. 2. In case of Ansakis spp. and Toxocara canis, enzymatic activity of these proteinases was considerably inhibited by Leupeptin and EDTA. For maximum enzymatic activity of above proteinases, it was required that cysteine residue of enzyme should be protected. And it was suggested that metallo type was contained in enzyme active site. Proteinase of Trichinella spiralis contained metallo type also. 3. Although partial purification was performed in Ansakis spp. and Toxocara canis, proteins maintaining enzymatic activity were identified as a circulating antigen. From SDS-PAGE and immunoblot, 25 kDa was presented in Ansakis spp.. Specific antigen of Toxocara cains was 110 kDa protein fraction. 55 and 42 kDa proteins were reacted with normal serum. Trichinella spiralis 60 kDa protein fraction was successfully purified from excretory materials in culture. As a result of immune-reaction with Trichinella spiralis infected serum, highly purified 60 kDa protein was maintained antigenicity until final purification step.

• 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.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.261-268
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• 1998

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin. TPCK and TLCK, and also by $Hg^{2+}$, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.

• International Journal of Oral Biology
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• v.34 no.2
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• pp.87-95
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• 2009

Porphyromonas gingivalis is a major etiologic agent of chronic periodontitis and cytokines produced by macrophages play important roles in the pathogenesis of periodontal diseases. In this study we investigated the cytokine response of phorbol myristate acetatedifferentiated THP-1 cells exposed to P. gingivalis. Compared with the prominent cell wall components of P. gingivalis (lipopolysaccharide and the major fimbrial protein FimA), live P. gingivalis stimulated much higher levels of cytokine production. In addition, whereas low multiplicity of infection challenges (MOI=10) of P. gingivalis 381 stimulated high levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and IL-1${\beta}$, high dose challenges with this bacterium (MOI = 100) resulted in a substantially diminished production of MCP-1 and IL-6. Moreover, high MOI P. gingivalis challenges achieved only low levels of induction of MCP-1 and IL-6 mRNA. The decreased production of MCP-1 and IL-6 appeared to be mediated by P. gingivalis proteases, because high MOI challenges with congenic protease mutant strains of this microorganism (MT10 and MT10W) did not result in a diminished production of MCP-1 and IL-6. Similar to its protease mutant strains, leupeptin (a protease inhibitor)- treated P. gingivalis at high doses induced high levels of MCP-1 production. To examine the mechanisms underlying the diminished production of MCP-1 by P. gingivalis proteases, the activation of mitogen-activated protein (MAP) kinases and NF-${\kappa}$B was compared between the 381 and MT10W strains. Whilst high doses of both 381 and MT10W similarly activated the three members of the MAP kinase family, the DNA binding activity of NF-${\kappa}$B, as revealed by gel shift assays, was greatly increased only by MT10W. Taken together, our data indicate that P. gingivalis stimulates the production of high levels of TNF-${\alpha}$, IL-1${\beta}$, IL-6, and MCP-1 but that high dose challenges with this bacterium result in a diminished production of MCP-1 and IL-6 via the protease-mediated suppression of NF-${\kappa}$B activation in THP-1 macrophagic cells.

• Parasites, Hosts and Diseases
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• v.31 no.2
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• pp.117-128
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• 1993

Ten axonic isolates of Trichomonns uaginolis were subcutaneously injected to the BALB/c mice in order to assess their pathogenicity by means of so-called "mouse assay" method. All the isolates revealed neutral and acid proteinase activities both in their Iysates and in culture media, but the specific activities of both proteinases in the severely pathogenic group were significantly higher than the mildly pathogenic group (p < 0.05). In the SDS-PAGE system in which the electrophoretic gels contained 0.4% gelatin as the substrate, five different handing patterns of trichomonal proteinases were detected, and the patterns were closely related with the pathogenicity of the isolates of T. vosinalis. All five bands might be regarded as cysteine proteinases group in the inhibitor assays. The cytotoxicity of the Iysates of T. vaginalis to the target Chinese hamster ovarian (CHO) cell line was also significantly different according to the pathogenicity of the isolates, and generally lower in the Iysates treated with cysteine proteinase inhibitors than in the control Iysates. In summarizing the results, it might be considered that the proteinases of T.vaginalis showing five electrophoretic banding patterns are closely related with the pathogenicity and cytotoxicity of the isolates of T. voginolis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.4
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• pp.282-290
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• 1992

Based on surplus production models using fishery data for the last 20 years, a stock assessment was conducted for the small yellow croaker in Korean waters. The maximum sustainable yields (MSY) from the Schaefer and Fox models were estimated to be 37,000 metric tons (mt) and 33,450 mt. Zhang's model using time-series biomass with instantaneous coefficients of fishing mortality (F) and using time-series biomass and catch yielded MSY estimates of 45,328 mt and 40,160 mt, respectively. A yield-per-recruit analysis showed that the current yield per recruit of about 20g with F= 1.11 $yr^{-l}$, where the age at first capture $(t_c)$ is 0.604, was much lower than the maximum possible yield per recruit of 43g. Fixing $t_c$ at the current level and reducing fishing intensity (F) from 1.11 $yr^{-l}$ to 0.4 $yr^{-l}$ yielded only a small increase in predicted yield per recruit, from 20 to 25g. However, estimated yield per recruit increased to 43g by increasing $(t_c)$ from the current age (0.604) to age three with F fixed at the current level. This age at first capture corresponded to the optimal length which was obtained from the $F_{0.1}$ method. According to the analysis of stock recovery strategies employing the Zhang model, the optimum equilibrium biomass $(B^*_{MSY})$ which produces the maximum yield could be achieved after approximately five years at the lower fishing intensity (F=0.5).