• BMB Reports
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• v.29 no.4
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• pp.380-385
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• 1996

Ovomucoid third domain is a serine proteinase inhibitor protein which consists of 56 amino acid residues. A fifty picosecond molecular dynamics (MD) simulation was carried out for ovomucoid third domain protein with 5 $\AA$ layer of water molecules. A comparison of main chain atoms in the MD averaged structure with the crystal structure showed that most of the backbone structures are maintained during the simulation. Investigation of the intramolecular hydrogen bondings indicated that most of the interactions between main chain atoms were conserved, whereas those between side chains were reorganized for the period of the simulation. Especially, the side chain interactions around the scissile bond of reactive site P1 (Met18) were found to be more extensive for the MD structures. During the simulation, hydrogen bonds were maintained between the side chains of Glu19 and Arg21 as well as those of Thr17 and Glu19. Extensive side chain interactions observed in the MD structures may shed light on the question of why protein proteinase inhibitors are strong inhibitors for proteinases rather than good substrates.

• The Korean Journal of Mycology
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• v.14 no.2
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• pp.101-107
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• 1986

The properties of carboxyl proteinase which was contained in Poria cocos (Schw.) Wolf were investigated by means of the purification with 0.65 ammonium sulfate saturation, DEAE cellulose and Sephadex G-75 gel filtration. This enzyme was found to hydrolyze only peptide bond between glutamyl-L-tyrosine of carbobenzoxy-L-glutamyl-L-tyrosine among the synthetic substrates of carbobenzoxy-L-glutamyl-L-tyrosine, hippuryl- L-phenylalanine and hippuryl-L-arginine. This enzyme was inhibited by $Zn^{+2},\;Fe^{+2},\;Ca^{+2},\;CN^{-1},\;P_2O_7^{-4}$ ions, but stimulated by $Hg^{+2}$ ion. Also, this enzyme was inhibited by organic compounds such as L-lysine, L-phenylalanine, hippuryl-L-phenylalanine, diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(P-nitrophenoxy)propane(EPNP). In particular, the activity was inhibited by L-lysine till 20 minutes of preincubation time rapidly, and by DAN in the presence of $Cu^{+2}$ ion more rapidly after 30 minutes than DAN in the absence of $Cu^{+2}$ ion. L-Lysine was found to be a competitive inhibitor and its $K_i$ value was determined to be 0.12 mmole by Dixon plot.

• Biomedical Science Letters
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• v.8 no.3
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• pp.161-165
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• 2002

Apoptosis can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of rat tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. I describe a new modified method for formalin-fixed, paraffin-embedded tissue sections, pretense pretreatment to permeate the tissue sections that involves an TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is acknowledged as a method of choice in the rapid identification and quantification of the apoptotic cell fraction in paraffin tissue preparations. TUNEL was performed without apoptosis and with apopotosis samples to each of the three concentrations of proteinase K (10, 25, 40 mg/ml) pretreatments. In this study, I show that chemical pretreatments of the tissue sections in proteinase K (25 mg/ml for 15 min at room temperature) considerably enhances the sensitivity of this nick end labelling technique.

• Parasites, Hosts and Diseases
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• v.40 no.2
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• pp.93-99
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• 2002

The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (slgA), IgG, and IgM. It also degraded $interleukin-1{\alpha}$ ($IL-l{\alpha}$) and $IL-l{\beta}$. Its activity was not inhibited by endogenous protease inhibitors, such as ${\alpha}$2-macroglobulin, ${\alpha}l-trypsin$ inhibitor, and ${\alpha}2-antiplasmin$. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthanoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection.

• Applied Biological Chemistry
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• v.33 no.4
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• pp.287-292
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• 1990

Eight(I through VII) of Bowman-Birk proteinase inhibitor have been isolated from soybean with DEAE-Sephadex A-50. Inhibitor VII was a typical BBTI, showing high cysteine content(17%/mole) ud low trypsin to chymotrypsin inhibiting activity(TIA/CIA= 1.0) with the independent reactive site to each enzyme. Dissociation constant of trypsin-BBTI and chymotrypsin-BBTl complexes were $9.17{\times}10^{-9}M$ and $5.14{\times}10^{-8}M$, respectively. Inhibitor Vll was extremely heat stable. Six hours heat treatment at $100^{\circ}C$ caused only 50% decrease in it's original inhibiting activity. Except inhibitor III,6 other isoinhibitors differed from a typical BBTI in TIA/CIA, values, ranging from 3 to 29.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.135-139
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• 1987

Aspergillus sp. MK-24 was cultured at 3$0^{\circ}C$ in the medium consisting of 2% glucose, 0.2% NaNO$_3$, 0.02% $K_2$HPO$_4$, 0.02% MgSO$_4$ 7$H_2O$, 0.02% KCl, and at initial pH of 5.0. The production of the inhibitor on venom proteinase reached to the maximum in 7 days. Sodium nitrate or potassium nitrate as a nitrogen source was favorable. The production of inhibitor was not affected by the addition of most of the inorganic salt used but depressed by lead, zinc, cobalt, mercuric or silver salts.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.381-385
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• 1996

Three distinct plasmids, with approximate molecular weights of 1, 4.5, and 33 megadaltons, were found in Lactococcus lactis subsp. lactis (L. lactis) ML8. Slow acid-producing mutants of L. lactis ML8, isolated by plasmid curing with acriflavine treatment, lacked the 33-megadalton plasmids. The plasmid-cured mutant showed lactose-negative (Lac) characteristics and the alteration of extracellular proteinase pattern. The possible involvement of extracellular proteinase with the 33-megadalton plasmid is highlighted in this research.

• Journal of Life Science
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• v.12 no.6
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• pp.752-758
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• 2002

This study was investigated on properties and thermostability of gelatin-degrading proteinases in the fruit of Actinidia chinensis (kiwifruit) for the industrial application. Three gelatin-degrading proteinases (PI, PII and PIII) were detected from the pulp of fruits. The molecular weights of these proteinases, PI, PII and PIII, were approximately 220 kD, 51 kD, and 26 kD respectively, on the basis of gelatin-containing SDS-PACE. The optimum pH of these proteinases ranged from 2.0 to 5.0 with a maximal activity at pH 4.0. These proteinases had a high sensitivity to E-64 and iodoacetate which are cysteine protease inhibitors, and required DTT, cysteine, and $\beta$-mercaptoethanol for their activities which are stimulators for cysteine proteases. These results indicate that these proteinases are cysteine proteinases and the proteinase PIII is actinidin (EC 3.4.22.14), based on the molecular weight and/or susceptibility against proteinase inhibitors. These proteinases were strongly activated by $Ca^{2+}$, $Mg^{2+}$ and $Mn^{2+}$, whereas strongly inhibited by Zn$^{2＋}$ and Hg$^{2＋}$. However, these proteinases have slightly different susceptibility against other cations ($Ca^{2+}$, $Cu^{2+}$, $Al^{3+}$, $Ca^{3+}$. The temperature stability of proteinase PIII was more stable than proteinases PI and PII. Moreover, proteinase PIII remained stable below $50^{\circ}C$ for 48hr, showing the residual activity above 75% of the enzyme activity.

• Development and Reproduction
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• v.10 no.4
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• pp.263-269
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• 2006

To evaluate the potentiality of industrial use of cysteine proteinase inhibitor (cystatin) of tilapia egg and serum stability of the tilapia cystatin on low temperature storage and heat treatment was studied. When the eggs were stored at $4^{\circ}C$ for 3 days the cystatin activity was not changed much, while the supernatant of egg homogenate lost its cystatin activity significantly, remaining only about 65% of initial activity. When the eggs and serum were subjected to repeated freeze at $-20^{\circ}C$ and thaw at room temperature once a day, the egg cystatin was decreased after 5 cycles of freeze and thaw. However the serum cystatin was not changed by the 5 times repetition of freeze and thaw. More than 80% of egg cystatin activity was remained when the egg was heated at $35^{\circ}C$ for 30 min, but less than 10% was remained when heated at $50^{\circ}C$. On the other hand, the serum cystatin was very resistant to heat, remaining about 74% after heating at as high as $80^{\circ}C$ for 30 min. In summary, the egg cystatin was more stable when stored as intact form of egg rather than as supernatant of homogenate when stored at refrigeration. Egg cystatin was relatively stable against repeated freeze-thaw, and serum was found to be more stable in cysteine proteinase inhibitory activity than egg. Egg cystatin was not very resistant to heat treatment, while serum cystatin was quite resistant to high temperature heat treatment. These results suggest that tilapia egg and serum, especially the serum, would be a useful source for cysteine proteinase inhibitor in surimi production.

• BMB Reports
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• v.41 no.11
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• pp.814-819
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• 2008

Interleukin-32 (IL-32) induces a variety of proinflammatory cytokines and chemokines. The IL-32 transcript was reported originally in activated T cells; subsequently, it was demonstrated to be abundantly expressed in epithelial and endothelial cells upon stimulation with inflammatory cytokines. IL-32 is regulated robustly by other major proinflammatory cytokines, thereby suggesting that IL-32 is crucial to inflammation and immune responses. Recently, an IL-32$\alpha$-affinity column was employed in order to isolate an IL-32 binding protein, neutrophil proteinase 3 (PR3). Proteinase 3 processes a variety of inflammatory cytokines, including TNF$\alpha$, IL-$1{\beta}$, IL-8, and IL-32, thereby enhancing their biological activities. In the current study, we designed four PR3-cleaved IL-32 separate domains, identified by potential PR3 cleavage sites in the IL-32$\alpha$ and $\gamma$ polypeptides. The separate domains of the IL-32 isoforms $\alpha$ and $\gamma$ were more active than the intrinsic $\alpha$ and $\gamma$ isoforms. Interestingly, the N-terminal IL-32 isoform $\gamma$ separate domain evidenced the highest levels of biological activity among the IL-32 separate domains.