• Bulletin of the Korean Chemical Society
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• v.4 no.3
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• pp.139-143
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• 1983

${\beta}$-N-Acetyl-D-glucosaminidase B was highly purified with the following sequence of steps; DEAE-cellulose, CM-cellulose, and Sephadex G-200 gel filtration chromatograpies. The specific activity of the purified ${\beta}$ -N-acetyl-D-glucosaminidase B was 2.2 units/mg protein with 12.9 % yield and 196.2 fold purity. The purified ${\beta}$-N-acetyl-D-glucosaminidase B showed single band on polyacrylamide gel electrophoresis. The final preparation of ${\beta}$ -N-acetyl-D-glucosaminidase B was completely free friom arylsulfatase and ${\beta}$-glucuronidase. ${\beta}$ -N-Acetyl-D-glucosaminidase B had pH optimum of 4.5 in 0.5 M sodium citrate buffer. The molecular weight of ${\beta}$-N-acetyl-D-glucosaminidase B was 133,000 by Sephadex G-200 gel filtration. The Km value of ${\beta}$-N-acetyl-D-glucosaminidase B using p-nitrophenyl-N-acetyl-${\beta}$-D-glucosaminide as substrate was 1.0 mM and $V_{max}$ was 0.014 ${\mu}$ mole/min. ${\beta}$-N-Acetyl-D-glucosaminidase B was stable at $55^{circ}C$ for 70 minutes. The crude ${\beta}$ -N-acetyl-D-glucosamiinidase in 70 % ammonium sulfate retained 93 % activity after 7 months storage at -$55^{circ}C$. Bovine serum albumin, sodium chloride, and phosphate activated ${\beta}$ -N-Acetyl-D-glucosaminidase B. N-Acetyl-D-glucosamine, ${\alpha}$-methyl-D-mannoside, and acetate inhibited ${\beta}$ -N-acetyl-D-glucosaminidase B.

• Environmental Mutagens and Carcinogens
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• v.21 no.2
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• pp.89-94
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• 2001

Recently, single cell gel electrophoresis, also known as comet assay, is widely used for the detection and measurement of DNA strand breaks in vitro and in vivo in many toxicological fields such as radiation exposure, human monitoring and toxicity evaluation. As well defined, comet assay is a sensitive, rapid and visual method for the detection of DNA strand breaks in individual cells. Briefly, a small number of damaged cells suspended in a thin agarose gel on a microscope slide were lysed, unwinded, electrophoresed, and stained with a fluorescent DNA binding dye. The electric current pulled the charged DNA from the nucleus such that relaxed and broken DNA fragments migrated further. The resulting images which were subsequently named for their appearance as comets, were measured to determine the extent of DNA damages. However, some variations could be occurred in procedures, laboratories's conditions and kind of cells used. Hence, to overcome and to harmonize these matters in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held with several topics including comet assay at Washington D.C. on March, 1999. In spite of some consensus in procedures and conditions in IWGTP, there are some problems still remained to be solved. In this respect, we attempted to set the practical optimal conditions in the experimental procedures such as lysis, unwinding, electrophoresis and neutralization conditions and so on. First of all, we determined optimal lysis and unwinding time by using 150 $\mu$M methyl methanesulfonate (MMS) which is usually used concentration. And then, we determined optimal positive control concentrations of benzo(a)pyrene (BaP) and MMS in the presence and absence of S9 metabolic activation system, respectively.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.39-44
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• 1999

Protein profiles of beef loin were constructed by comparing two-dimensional gel electrophoresis patterns of the proteins from different growth stages of Hanwoo, Korean cattle. Proteins from the lean muscle of 0, 6, 12 and 24 months old Hanwoo were separated by isoelectric focusing (IEF) on 16 cm tube gels, and further processed second dimensionally by 12% SDS-polyacrylamide gel electrophoresis (PAGE) using $18{\times}20$ cm gel. Proteins with pI values ranging 3.0 to 9.0 and molecular weight of 15 to 100 kDa could be clearly detected on gel by silver staining. Interestingly, many of the proteins significantly increased and decreased during growth happened to be low molecular ones. To isolate the increased proteins, the soluble proteins were obtained from the tissue by 1% Triton X-100 extraction, then, fractionated by 30% and 50% ammonium sulfate. The isolation condition of each particular protein was determined. According to the conditions, two of the increased proteins were isolated, and transferred to PVDF membrane and microsequenced.

• Molecules and Cells
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• v.20 no.2
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• pp.271-279
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• 2005

Although macrophages are an important first line of cellular defense, they are unable to effectively kill phagocytosed C. albicans. To determine the physiological basis of this inability, we investigated the alterations of macrophage proteins caused by C. albicans infection. Since the formation of C. albicans hyphae caused cell death, proteins were prepared 3 h after infection and examined by two-dimensional gel electrophoresis (2-DE). The most prominent changes were in glycolytic enzymes, which could have caused energy depletion of the infected cells. Also changed were proteins involved in maintenance of cellular integrity and NO production. Treatment of the macrophages with either cytochalasin D or taxol did not alter their inability to kill C. albicans. Our results indicate that multiple factors contribute to cell death as the pathogenic form of C. albicans becomes fully active inside macrophage cells.

• Journal of the Korea Organic Resources Recycling Association
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• v.1 no.1
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• pp.115-125
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• 1993

In Euglena gracilis arginine deiminase was located in the mitochondrial matrix. The highly purified enzyme required $Co^{2+}$ for the enzyme reaction with the $K_m$ value of 0.23 nM, and its optimum pH was 9.7 to 10.3. The molecular weight of the native enzyme protein was 87,000 by gel filtration, and SDS-acrylamide gel electrophoresis showed that the enzyme consisted of two identical subunits with a molecular weight of 48,000. Euglena arginine deiminase was inhibited by sulfhydryl inhibitors, indicating that a sulfhydryl group is involved in the active center of the enzyme. It exhibited negative cooperativity in binding with arginine. $L-{\alpha}-amino-{\beta}-guanidino-propionate$, D-arginine, and L-homoarginine strongly inhibited the enzyme while ${\beta}-guanidinopro-pionate$, ${\gamma}-guanidinobutyrate$, and guanidinosuccinate did not. Considerable inhibition was also observed with citrulline and ornithine. We discuss the effects of the unique properties of the Euglena arginine deiminase on the regulation of arginine metabolism in this protozoon.

• Microbiology and Biotechnology Letters
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• v.24 no.2
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• pp.197-205
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• 1996

The $\beta$-xylosidase was purified 99- fold from the culture supernatant of Pseudo onas sp. CB-33 by ammonium sulfate precipitation, PEI precipita- tion, DEAE-Sephadex column chromatography, Sephadex G-75 gel filtration chromatography and preparative disc gel electrophoresis. Molecular weight of the enzyme was estimated to be 44,000 by SDS polyacrylamide gel electrophoresis. The enzyme has a pH optimum for activity at 7.0 and is stable over pH 6.5-9.0. The optimal temperature of the enzyme was 45$\circ$C, and its enzymatic activity was completely inactivated at 55$\circ$C for 30 min. Km value of the enzyme for p-nitrophenyl-$\beta$-D-xylopyranoside was calculated to be 4.6 mM. The effect of various reagents on the $\beta$-xylosidase activity was investigated. The enzyme activity was completely inhibited by Hg$^{2+}$, Cu$^{2+}$ and Zn$^{2+}$. The $\beta$-xylosidase was inactivated by tryptophan-specific reagent, N-bromosuccinimide and tyrosine-specific reagent, iodine. The enzyme could degrade xylo-oligosaccharides to xylose and the enzyme was competitively inhibited by xylose. The $\beta$-xylosidase and endoxylanase from Psedomonas sp. CB-33 hydrolized xylan synergically. The purified enzyme also showed $\alpha$-L-arabinofuranosidase activity.

• Microbiology and Biotechnology Letters
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• v.21 no.6
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• pp.588-593
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• 1993

Xylanase(1, 4-beta-D-xylan xylanohydrolase` EC 3.2.1.8) II was purified from Penicillium verruculosum by using the techniques of two anion exchange chromatographies, and gel filtration. The molecular weight of this enzyme was about 22, 000 as determined by SDS-electrophoresis. The enzyme showed hydropytic activity toward xylan but did not catalyze hydrolysis of Rho-nitrophenyl-beta-D-xylopyranoside, Rho-nitrophenyl-beta-D-glucopyranoside, Rho-nitrophenyl-beta-D-cellobiopyranoside, and celluloses such as Avicel, cotton, filter paper, carboxymethylcellulose.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.63-68
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• 2005

Cloned calves derived from somatic cell nuclear transfer (SCNT) have been frequently lost by sudden death at 1 to 3 month following healthy birth. To address whether placental anomalies are responsible for the sudden death of cloned calves, we compared protein patterns of 2 placentae derived from SCNT of Korean Native calves died suddenly at two months after birth and those of 2 normal placentae obtained from AI fetuses. Placental proteins were separated using 2-Dimensional gel electrophoresis. Approximately 800 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis of Malanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, 8 spots were identified to be up-regulated proteins and 24 spots to be down-regulated proteins in SCNT placentae, among which proteins were high mobility group protein HMG1, apolipoprotein A-1 precursor, bactenecin 1, tropomyosin beta chain, $H^+-transporting$ ATPase, carbonic anhydrase II, peroxiredoxin 2, tyrosine-rich acidic matrix protein, serum albumin precursor and cathepsin D. These results suggested that the sudden death of cloned calves might be related to abnormal protein expression in placenta.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.253-260
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• 2007

To examine the differential protein expression pattern in the 11.5 day post-coitus (dpc) and 18.5 dpc placenta of mouse, we have used the global proteomics approach by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The differential protein patterns of 3 placentae at the 11.5 dpc and 18.5 dpc from nature mating mice were analyzed. Proteins within isoelectric point range of $3.0{\sim}10.0$, separately were analyzed in 2DE with 3 replications of each sample. A total of approximately 1,600 spots were detected in placental 2-D gel stained with Coomassie-blue. In the comparison of 11.5 dpc and 18.5 dpc placentae, a total of 108 spots were identified as differentially expressed proteins, of which 51 spots were up-regulated proteins such as alpha-fetoprotein, mKIAA0635 protein and transferrin, annexin A5, while 48 spots were down-regulated proteins such as Pre-B-cell colony-enhancing factor l(PBEF), aldolase 1, A isoform, while 4 spots were 11.5 dpc specific proteins such as chaperonin and Acidic ribosomal phosphoprotein P0, while 3 spots were 18.5 dpc specific proteins such as aldo-keto reductase family 1, member B7 and CAST1/ERC2 splicing variant-1. Most identified proteins in this analysis appeared to be related with catabolism, cell growth, metabolism and regulation. Our results revealed composite profiles of key proteins involved in mouse placenta during pregnancy.

• Proceedings of the Zoological Society Korea Conference
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• 2003.08a
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• pp.176.1-176
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• 2003

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