• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.275-281
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• 2010

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of Mil stage.

• 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.

• Journal of Life Science
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• v.12 no.5
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• pp.528-534
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• 2002

A kypsin inhibitor was isolated and purified from Mung bean (Vigna radiata L. wilczek) which has been used as a galenic and traditional food. In addition, we evaluated the pharmacological effect of the mung bean trypsin inhibitor (MBTI) using septic shock induced guinea pig model. Purification was carried out by Sephadex G-50 gel filtration, DEAE-cellulose ion exchange chromatography, and trypsin affinity column. The molecular weight of MBTI was estimated to be about 8,000 Da by 20% SDS-PACE under reducing condition. The chemically determined partial amino acid sequences of the purified MBTI perfectly coincide with those of previously reported MBTI which is BBI type trypsin inhibito. (Bowman-birk inhibitor type). These results suggest that the purified MBTI is authentic. Hypotension shock was prevented by the pretreatment of the MBTI (10 mg/kg of the body weight) on the septic shock guinea pig model caused by psedomonal elastase.

• KSBB Journal
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• v.13 no.2
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• pp.195-202
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• 1998

The cholesterol oxidase was purified from the culture broth of Rhodococcus sp. 3T6-5Mj strain by procedures involving filtration, acetone precipitation, DEAE-Sephadex A-50, and cholesterol affinity column chromatography with a recovery of 15% to specific activity of 25.6 units/mg. The molecular weight of the enzyme was estimated to be 52,000 daltons by SDS-PAGE. Optimum pH and temperature for the enzyme activity were approximately pH 7.0 and $50^{\circ}C$ respectively. The Michaelis constant (Km) for cholesterol was found to be $3.2{\times}10^{-4}$ M. The enzyme showed a high substrate specificity for $3{\beta}$-hydroxysterols and the relative oxidation rates were 100% for cholesterol, 89% for campesterol, 55% for stigmasterol, etc. Amino acid analysis showed that the enzyme protein was composed of 440 amino acid residues without cystein and tryptophan.

• Journal of Microbiology
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• v.39 no.4
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• pp.279-285
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• 2001

Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1:K1 was isolated from the environment and identified . A polymerase chain reaction assay revealed that this strain harbored both the tdh and the genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6h of culti-vation at 37$\^{C}$ under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85kDa, 59kDa, 41kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255kDa. The purified urease was stable at pH 7.5 and the opeimal pH in HEPES buffer was 8.0 The enzyme was stable at 60$\^{C}$ for 2 h with a residual activity of 32% . The addition of 10$\mu$M if NiCl$_2$maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD$\_$50/(median toxic dose) of the purified urease was 2.5$\mu\textrm{g}$/ml on human leukemia cells.

• BMB Reports
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• v.28 no.2
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• pp.118-123
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• 1995

Biodegradative threonine dehydratase was purified to homogeneity from Serratia marcescens ATCC 25419 by streptomycin sulfate treatment, Sephadex G-200 gel filtration chromatography followed by AMP-Sepharose 4B affinity chromatography. The molecular weight of the purified enzyme was 118,000 by fast protein liquid chromatography using superose 6-HR. The enzyme was determined to be a homotetrameric protein with subunit molecular weights of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by ${\alpha}-Keto$ acids and their derivatives such as ${\alpha}-ketobutyrate$, pyruvate, glyoxlyate, and phosphoenol pyruvate, but not by ${\alpha}-aminobutyrate$ and ${\alpha}-hydroxybutyrate$. The inhibition of the enzyme by pyruvate and glyoxylate was observed in the presence of AMP. The inhibitory effect of glyoxylate was decreased at high enzyme concentration, whereas the inhibition by pyruvate was independent of the enzyme concentration. The kinetics of inhibition of the enzyme by pyruvate and glyoxylate revealed a noncompetitive and mixed-type inhibition by the two inhibitors with respect to L-threonine and AMP, respectively.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.141-141
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• 1993

말징버섯 Calvatia craniformis의 culture broth 40 liter를 여과하여 얻은 균사채를 열수 추출하여 진한 갈색 건조분말(Fr. A) 13.1g을 분리하였다. Fr. A에 대하여 DEAE-cellulose ion chromatography를 시행하여 중성분획인 흰색 분말 (Fr. B) 2.50g을, 산성분획인 갈색 분말 (Fr. C) 3.50g을 각각 분리하였다. Fr. B 500mg에 대하여 Sepharose CL-4B gel filtration chromatography를 시행하여 흰색분말 Fr. D(Calvatan) 350mg을 분리하였다. Calvatan 350mg에 대하여 Con A-Sepharose 4B affinity chromatography를 적용하여 미흡착 분획인 Fr. F($\alpha$-form)와 흡착 분획인 Fr. E ($\beta$-form)로 정제하였다. 항암작용의 기전을 구명하고자 마우스에 대하여 면역학적 실험을 시행하였다. macrophage의 활성에 대한 영향을 조사하였던 바, 투여군의 활성화된 macrophage에 의해 유리되는 superoxide anion의 양은 대조군에 비해 1.4배 증가하였고 Calvatan 투여군의 용혈반 형성세포(PFC)는 대조군에 비해 3.1배 증가하였다. 화학 분석에 의해, Calvatan은 다당체 87.2%, 단백질 1.8% 및 hexosamine 1.3%로 구성되어 있었다. 따라서 항암성 분획들은 protein-bound polysaccharide임을 알 수 있었다. 또한 각 분획의 다당체를 구성하고 있는 단당류는 glucose, galactose, mannose 및 xylose 였으며 단백질 부분은 16종의 아미노산으로 구성되어 있었다. IR 스펙트럼은 3300-3400 $cm^{－1}$에서 0-H stretching frequency, 2900 $cm^{－1}$ 에서 C-H stretching frequency, 1630 $cm^{－1}$ 에서 C-0 stretching frequency. 1000-1100 $cm^{－1}$에서 C-H 및 C-0 bending frequency를 나타내는 다당체의 전형적인 특성을 보여주었다.을 보여주었다.

• Korean Chemical Engineering Research
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• v.52 no.1
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• pp.98-105
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• 2014

Lysozyme amounts to 0.3% in egg white and functions as an agent of cell lysis and activator of tissue reconstruction. Ion exchange chromatography is the most useful method of separation among affinity chromatography, ion exchange chromatography, and ultra-filtration. The aim of present study is to find the optimum pH and temperature for the separation of lysozyme in egg white within cation exchange gel filled glass column. And we compared results of experiments with those of simulations. Phosphate buffer was used, and pH and temperature were varied as 5~7 and $25{\sim}40^{\circ}C$ respectively. RP-HPLC was the tool for the retention time identification and quantitative analysis of lysozyme. OriginPro 8 measured the peak area of lysozyme chromatogram and quantified the eluted lysozyme. Largest amount of lysozyme was separated under the conditions of pH 5 and T $25^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.335-342
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• 1988

An anion exchange chromatography was employed for the purification of mouse monoclonal antibodies from ascitic fluid and in vitro cultivation media. After cultivation of hybridomas, Alps 25-3, HCGK, A4W, and KW, producing IgG1, the culture supernatants were harvested by centrifugation, precipitated with 50-60％ ammonium sulfate, and dialyzed against 0.025 M Tris-HCI buffer (pH 8.2). Then the dialyzed samples were loaded into a DEAE-Trisacryl M anion exchange column. Monoclonal antibodies bound to the DEAE-Trisacryl M were eluted with 0.025 M Tris-HCI buffer (pH 8.2) containing 30-40 mM NaCl. In ammonium sulfate precipitation, the recovery of the monoclonal antibody was shown to be 90％ and 84％ from in vitro culture media containing 10％ and 2％ fetal bovine serum, respectively. On the other hand, the pretreatment by ultrafiltration enhanced the yield up to 91％ whereas the purity was lower than that by ammonium sulfate treatment. Subsequently, in the DEAE-Trisacryl M chromatographic separation, the purities and recoveries of all the monoclonal antibodies from both the in vitro culture supernatants and ascitic fluids were 70-80％ and 65％ respectively. The monoclonal antibody, Alps 25-3 could be further purified with a purity of 95％ through an immunoadsorbent chromatography.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.6
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• pp.964-972
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• 1994

The 4.3-kb gene coding for L-lactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment , DEAE-Sephadex, and NAD++ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200 . The molecular weight of the purfied enzyme was estimated to be about 35, 000 and 140, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. indicating that the enzyme is composed of four identical subunits. THe enzyme for pyruvate reduction and lactate oxdiation was stable at 60 and 75$^{\circ}C$ for 30 min, and the optimal temperatures for both reactions were 60 and 7$0^{\circ}C$, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme of pyruvate reduction was table between pH 5 and 7. more than 90% of enzyme activity was lost at 1mM FeSO4 and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1, 6-bisphosphate.