• YAKHAK HOEJI
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• v.37 no.2
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• pp.149-157
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• 1993

Protein methylase inhibitor which is a modulator of biological methylation has been purified and characterized from porcine liver soluble fraction by cell fractionation, Sephadex G25 chromatography, reverse phase HPLC, size exclusion HPLC. The results are summarized as follows. 1) The purified inhibitor shows apparent homogeneity, as judged by HPLC. 2) A molecular weight of the purified inhibitor which is composed of 18 amino acid residues is about 1,400 daltons. 3) A single absorption peak of ultraviolet spectrum was observed at 260nm. 4) The inhibitor was not inactivated by heating at $100^{\circ}C$ until 60min. and its activity was not influenced by treatment with digestive enzymes, such as trypsin, pepsin, pronase, chymotrypin, lysozyme, DNase, and RNase. 5) The purified inhibitor inhibited protein rnethylase I, II, III and phospholipid methyltransferase activities. 6) The purified inhibitor inhibited noncompetitively protein methylase II from porcine liver, spleen, and testis. 7) The $K_{i}$ values for protein methylase II from porcine liver, spleen, and testis were 300nM, 250nM, 297nM, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.430-436
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• 2010

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

• Journal of Life Science
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• v.25 no.2
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• pp.180-188
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• 2015

The bacterial strain isolated from Kimchi showed antibacterial activity against Micrococcus luteus IAM 1056. The selected strain was identified as Lactococcus lactis by 16S rRNA nucleotide sequence analysis and named as Lactococcus sp. KD 28. The treatment of culture supernatant with proteinase K removed antibacterial activity, indicating its proteinaceous nature, a bacteriocin. This bacteriocin was sensitive to hydrolytic enzymes such as ${\alpha}$-chymotrypsion, trypsin, proteinase K, lipase, ${\alpha}$-amylase and subtilisin A. The bacteriocin was highly thermostable and resistant to heating at $80^{\circ}C$ for up to an hour but 50 % of the total activity was remained at $100^{\circ}C$ for 30 min. The pH range from 2.0 to 8.0 had no effect on bacteriocin activity and it was not affected by solvents such as acetonitrile, isopropanol, methanol, chloroform and acetone up to 50% concentration. The bacteriocin showed antibacterial activity against M. luteus IAM 1056, Lactobacillus delbrueckii subsp. lactis KCTC 1058, Enterococcus faecium KCTC 3095, Bacillus cereus KCTC 1013, B. subtilis KCTC 1023, Listeria ivanovii subsp. ivanovii KCTC 3444, Staphylococcus aureus subsp. aureus KCTC 1916, B. megaterium KCTC 1098 and B. sphaericus KCTC 1184. The bacteriocin was purified through ammonium sulfate concentration, SP-Sepharose chromatography and RP-HPLC. The molecular weight was estimated to be about 3.4 kDa by tricine-SDS-PAGE analysis.

• Journal of Dairy Science and Biotechnology
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• v.17 no.1
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• pp.1-10
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• 1999

A bacteriocin produced by Lab. acidophilus GP4A isolated from fecal contents of pig was characterized. Lab. acidophilus GP4A produced a heat-stable and pH-resistant bacteriocin, which was hydrolyzed by trypsin and pepsin and active against various microorganisms. Lab. acidophilus GP4A produced bacteriocin at maximum rate when grown in MRS broth(pH 6.5${\sim}$7.5) at$37^{\cric}C$ or $40^{\cric}C$. The bacteriocin produced by Lab. acidophilus GP4A inhibited the growth of Lactobacillus delbrueckii subsp. lactis 4794 in early logarithmic phase. The bacteriocin was purified by ammonium sulfate precipitation and Octyl sepharose CL-4B column chromatography. The purification resulted in a final yield of 21.7% and a 13.6-fold increase in the specific activity.

• Toxicological Research
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• v.17
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.461-468
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• 2016

In recent years, various naturally occurring defence peptides such as plectasin have attracted considerable research interest because they could serve as alternatives to antibiotics. However, the production of plectasin from natural microorganisms is still not commercially feasible because of its low expression levels and weak stability. A tandemly arrayed plectasin gene (1,002 bp) from Pseudoplectania nigrella was generated using the isoschizomer construction method, and was inserted into the pPICZαA vector and expressed in Pichia pastoris. The selected P. pastoris strain yielded 143 μg/ml recombinant plectasin (Ple) under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Ple was estimated by SDS-PAGE to be 41 kDa. In vitro studies have shown that Ple efficiently inhibited the growth of several gram-positive bacteria such as Streptococcus suis and Staphylococcus aureus. S. suis is the most sensitive bacterial species to Ple, with a minimum inhibitory concentration (MIC) of 4 μg/ml. Importantly, Ple exhibited resistance to pepsin but it was quite sensitive to trypsin and maintained antimicrobial activity over a wide pH range (pH 2.0 to 10.0). P. pastoris offers an attractive system for the cost-effective production of Ple. The antimicrobial activity of Ple suggested that it could be a potential alternative to antibiotics against S. suis and S. aureus infections.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.5
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• pp.478-483
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• 2011

An antimicrobial material was purified from the acidified whole body extract of the jellyfish Nemopilema nomurai by using C18 reversed phase and cation-exchange high performance liquid chromatography (HPLC). Whole body extract and the purified compound (JAP-1) showed potent antimicrobial activities against a wide range of microorganisms including Escherichia coli D31, Bacillus subtilis, Streptococcus iniae and Candida albicans, without significant hemolytic activity. Treatment of JAP-1 with trypsin completely abolished all antibacterial activity against Bacillus subtilis, suggesting that JAP-1 is likely to be a proteinaceous antibiotic. The molecular weight of JAP-1 was determined to be 680.10 Da by MALDI-TOF mass spectroscopy.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1393-1400
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• 2009

A bacteriocin-producing strain PI80 was isolated from the gut of Penaeus indicus (Indian white shrimp) and identified as Streptococcus phocae PI80. The bacteriocin was purified from a culture supernatant to homogeneity as confirmed by Tricine SDS-PAGE. Reverse-phase HPLC analysis revealed a single active fraction eluted at 12.94 min, and MALDI-TOF mass spectrometry analysis showed the molecular mass to be 9.244 kDa. This molecular mass does not correspond to previously described streptococcal bacteriocins. The purified bacteriocin was named phocaecin PI80 from its producer strain, as this is the first report of bacteriocin production by Streptococcus phocae. The bacteriocin exhibited a broad spectrum of activity and inhibited important pathogens: Listeria monocytogenes, Vibrio parahaemolyticus, and V. fischeri. The antibacterial substance was also sensitive to proteolytic enzymes: trypsin, protease, pepsin, and chymotrypsin, yet insensitive to catalase, peroxidase, and diastase, confirming that the inhibition was due to a proteinaceous molecule (i.e., the bacteriocin), and not due to hydrogen peroxide or diacetyl. Phocaecin PI80 moderately tolerated heat treatment (up to $70^{\circ}C$ for 10 min) and resisted certain solvents (acetone, ethanol, and butanol). A massive leakage of $K^+$ ions from E. coli $DH5\alpha$, L. monocytogenes, and V. parahaemolyticus was induced by phocaecin PI80, as measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES). Therefore, the results of this study show that phocaecin PI80 may be a useful tool for inhibiting L. monocytogenes in seafood products that do not usually undergo adequate heat treatment, whereas the cells of Streptococcus phocae PI80 could be used to control vibriosis in shrimp farming.

• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.99-108
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• 1996

3종의 빙핵세균 Peudomonas syringae 8401, Pseudomonas fuorescens 8701, Erwinia herbicola 8701의 세포 외막으로부터 아무런 변성제도 사용치 않고 sucrose density gradient centrifugation, Sephacryl gel filtration chromatography, DEAE-cellulose ion exchange chromatography, non-denaturing buffer를 이용한 PAGE, electroelution, SDS-PAGE를 통해 빙핵활성 단백질을 고도로 정제할 수 있었다. P. suringae와 P. fluorescens에서는 각각 3종류(155 kD, 75 kD, 50 kD)의 빙핵활성 단백질이, E. herbicola에서는 155 kD를 제외한 2종류(75 kD, 50 kD)의 빙핵활성 단백질은 이 연구를 통해 처음 밝혀진 것으로 , 지금까지 보고된 빙핵활성 단백질(150 KD 이상)보다는 훨씬 작은 것이다. 이는 빙핵활성을 나타내는 단백질의 기본단위는 이 실험의 결과만에 의하면 최대 50 kD임을 시사한다. 이들 단백질은 그 유래된 세균의 종류나 또는 단백질 분자량의 크기에 관계없이 모두 -5.5~7.5$^{\circ}C$에서 물을 동결시키는 높은 빙핵활성을 갖고 있었다. 이는 지금까지 보고된 어느 정제단백질의 빙핵활성보다 높은 것이다. 정제된 단백질의 빙핵활성은 trypsin 처리에 의해 상실되었고, pH6~8범위에서는 안정하였으며, pH5이하, pH9이상에서는 활성을 상실하였다. 보존온도에 대한 영향은 3$0^{\circ}C$이상이 되면 점차 활성이 감소하는 경향을 보이다 37$^{\circ}C$이상에서는 활성이 완전히 상실되었다. 금속이온으로서 Hg\ulcorner이온과 SDS에 의해 활성이 상실되었으나 phosphatidylinositol의 첨가에 의해서는 활성이 약간 증가(-1$^{\circ}C$)하였다.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.799-803
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• 2005

In the course of screening anti-dementia agents from natural products, two $\beta$-secretase (BACE1) inhibitors were isolated from the ethyl acetate soluble fraction of Sanguisorbae Radix by the activity-guided purification using silica gel, Sephadex LH-20, and RP-HPLC. They were identified as 1,2,3-trigalloyl-4,6-hexahydroxydiphenoyl-$\beta$-D-glucopyranoside (Tellimagrandin II, 1) and 1,2,3,4,6-pentagalloyl-$\beta$-D-glucopyranoside (2) and were shown to non-competitively inhibit $\beta$-secretase (BACE1) with the $IC_{50}$ values of $3.10{\times}10^{-6}M\;and\;3.76{\times}10^{-6}M$, respectively. The Ki values of 1 and 2 were $6.84{\times}10^{-6}M\;and\;5.13{\times}10^{-6}M$. They were less inhibitory to asecretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.