• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.2
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• pp.83-88
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• 2005

Protease inhibitors were purified from the eggs of Alaska pollock (Theragra chalcogramma) using the following purification steps: ammonium sulfate precipitation, ion exchange, gel permeation, and high performance liquid chromatographies (HPLC). The protease inhibitor from the heated eggs of Alaska pollock was not as well purified. In addition, the heated eggs showed lower specific inhibitory activity than the unheated eggs. The purification yields after ammonium sulfate precipitation, ion exchange, and gel permeation chromatographies were 22.7$\%,\;15.3\%$,and $4.4\%$, respectively. There were two kinds of protease inhibitors on the gel permeation chromatography pattern Their molecular weights were estimated to be 66,700 and 16,000 Da, respectively. Both were classified as a cysteine protease inhibitor because of the existence of inhibiting papain, which is one of cysteine proteases.

• BMB Reports
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• v.31 no.4
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• pp.364-369
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• 1998

Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its $NH_{2}-terminus$ was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.

• Parasites, Hosts and Diseases
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• v.38 no.3
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• pp.195-199
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• 2000

The 27 kDa cathepsin L-like cysteine protease of Spirometra erinocei plerocercoid is known to play an important function in tissue penetration, nutrient uptake and immune modulation in human sparganosis. In the present study, the expression of this enzyme was examined at different developmental stages of S. erinacei including immature egg, coracidium, plerocercoid in tadpole and rat, and adult Proteolytic activity against carboxybenzoyl-phenylalanyl-arginyl-7-amino-4-rnethylcournarin was do tooted in the extracts of coracidia and plerocercoid while no activity was observed in those of immature egg and adult. The specific activity in coraridial extracts was lower than that in the plerocercoid. Reverse transcription-polymerase chain reaction and Northern biol analysis demonstrated that the gene was expressed in the coracidium and plerocercoid but not in immature egg and adult. These results suggest that the 27 kDa cysteine protease is only expressed in the stages involving active migration of the parasite in the host tissue.

• Applied Biological Chemistry
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• v.32 no.2
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• pp.116-125
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• 1989

Bowman-Birk type protease inhibitors of soybeans were purified with gel filtration on Sephadex G-75. Significant differences were found in the content and in the electrophoretic patterns of the purified protease inhibitors. Ten among fourteen electrophoretic bands appeared as protease inhibitors. The chymotrypsin and trypsin inhibiting activities in soybeans showed three and four fold variation respectively. And the cultivars with higher chymotrypsin inhibiting activities seemed to have higher cysteine contents.

• Parasites, Hosts and Diseases
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• v.38 no.2
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• pp.95-97
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• 2000

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4 guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimuf westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westemani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.

• Parasites, Hosts and Diseases
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• v.43 no.1 s.133
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• pp.33-37
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• 2005

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EON) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.

• Journal of Life Science
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• v.9 no.1
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• pp.58-63
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• 1999

Highly metastatic mouse T-lymphoma CS21 cells can grow in vitro when cocultured with CA12 lymph node stromal cells, but they undergo apoptotic cell death when separated from CA12 stromal cells. It has been found that cysteine and interleukin-7(IL-7) as antiapoptotic soluble factors that produced by CA12 stromal cells. In this study, we report that an ICE family protease is activated in CS21 cells when separated from CA12 stromal cells and cultured alone. Enzyme purification using an avidin affinity column revealed that the involved cysteine protease possessed caspase3-like death protease activity. In addition, when IL-7 was added to CS21 cell culture, the protease activity could not be detected during partial purification of the enzyme. Taken together, these results strongly suggest that the caspase3-like protease activation is suppressed by IL-7 as an antiapoptotic factor that leads to abrogation of apoptosis execution.

• Food Science and Biotechnology
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• v.16 no.2
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• pp.294-298
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• 2007

The actinidin (EC 3.4.22. 14) found in kiwifruit is a cysteine protease. In order to obtain the actinidin gene from the Chinese wild kiwifruit, primers were designed on the basis of the actinidin gene of Actinidia deliciosa, the New Zealand kiwifruit. The 1.2 kb DNA fragment was acquired from the total RNAs of Chinese wild kiwifruit via reverse transcription polymerase chain reaction (RT-PCR), and its DNA sequence was analyzed. Its sequence was determined to share 98.4% homology with the actinidin gene of A. deliciosa. In order to verity the actinidin gene isolated from the Chinese wild kiwifruit in Escherichia coli, the mature gene was amplified via PCR and expressed in E. coli under the control of the T7lac promoter. The actinidin was expressed in E. coli as inclusion bodies, which were solubilized with urea and refolded. The protease activity of the refolded protein was approximately twice as high as that of E. coli BL2l (DE3).

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.87.1-87.1
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• 2007

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• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.139-142
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• 1997

Cleaving host immunoglobulins is a well known mechanism of evading host immune reactions exploited by helminth parasites, Secreted cysteine proteases of helminth are a part of enzymes cleaving host IgG. Porogonimw westemani produces at least six different species of the cysteine protease in its developmental stages. This study was undertaken to evaluate comparatively the activities against human IgG by the different enzymes. When the metacercariae, which secrete 27 and 28 kDa cysteine proteases, were incubated in human IgG solution, IgG was degraded at its hinge region. Further incubation resulted complete hydrolysis. From 4-week and 7-week old juveniles and 16-week old adults of p. westemani, five different enzymes at 15, 17. 27 28 and 53 kDa have been purified, if the enzyme with the same molecular mass is regarded to be identical. In cleaving human IgG, each cysteine protease exhibited decreasing activities with age.