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An efficient and reliable electroelution method from SDS-PAGE: Identification of a 31 kDa protein in the postsynaptic density fraction as adenine nucleotide translocator 1 (SDS-겔로부터 효율적인 단백질 분리방법과 31 kDa 연접후치밀질 단백질의 동정)

  • Jung, Jae-Seob;Cho, Sun-Jung;Shin, Seung-Chul;Jin, Ing-Nyol;Jung, Yong-Wook;Ko, Bok-Hyun;Moon, Il-Soo
    • Journal of Life Science
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    • v.12 no.5
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    • pp.555-560
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    • 2002
  • The molecular composition of the postsynaptic density (PSD) is largely hon. In this report, an electroelution protocol was demonstrated to be used for efficient isolation of PSD proteins with diverse molecular sizes. Using this protocol, a 31 kDa protein in the 1% n-octyl glucoside-insoluble PSD fraction (termed as PSD31) was purified from SDS-gels, and internal peptides were determined for amino acid sequences. The amino acid sequences of the PSD31 were highly homologous with the adenine nucleotide translocator 1 (ANTI). The association of ANTl with PSD suggests presence of a mechanism in synapses for releasing adenosine nucleotides into the extracellular space.

Cloning of a Alkaline Protease Gene from Xanthomonas sp. YL-37 (Xanthomonas sp. YL-37의 Alkaline Protease 유전자의 클로닝)

  • 이대희;김수경;이승철;윤병대;황용일
    • Microbiology and Biotechnology Letters
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    • v.23 no.2
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    • pp.145-149
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    • 1995
  • For the purpose of developing a new biodegradable detergent, we have isolated a gene encoding wide-range temperature applicable alkaline protease from Xanthomonas sp. YL-37 (Lee et al., 1994, Kor. J. Appl. Microbiol. Biotechnol.). An alkaline protease gene was isolated from the gene bank that was prepared from the chromosomal DNA of Xanthomonas sp. YL-37. From the results of agarose gel electrophoresis and a restriction enzyme mapping, a 2.7 kb DNA fragment containing the alkaline protease gene was inserted in the plasmid pUC9. Extracellular activity of a clone having alkaline protease gene was detected on SDS-polyacrylamide gel with activity staining assay. The molecular weight of alkaline protease was determined to be about 64 kDa from 11% SDS-PAGE analysis. Alkaline protease activity, produced from E. coli which harboring the plasmid, showed no difference at reaction temperature 20, 30 and 40$\circ$C, respectively. This result showed that alkaline protease produced from E. coli harboring the plasmid was apparently the same as that of Xanthomonas sp. YL-37.

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Nonspecific Association of a 17 kDa Isoform of the Myelin Basic Protein with the Postsynaptic Density Fraction

  • Moon, Il-Soo
    • BMB Reports
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    • v.33 no.3
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    • pp.276-278
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    • 2000
  • The postsynaptic density (PSD), a large protein complex beneath the postsynaptic membrane, is notorious for its 'stickiness'. In order to understand the molecular composition of the PSD fraction, a 17 kDa protein band was isolated by electroelution from SDS-geis, and its partial amino acid sequence was determined from HPLC-purified tryptic peptides of the protein. Surprisingly, the amino acid sequence was identical to that of the previously reported 17 kDa isoform of the myelin basic protein (MBP), an essential protein in CNS myelin formation. Since the protein band represented ~2% of the total proteins in the 1 % n-octyl glucoside-insoluble PSD fraction, these results indicate that a significant amount of the 17 kDa isoform of MBP is tightly associated with the PSD during preparation of the PSD fraction.

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Purification and Characterization of Polyphenol Oxidase in the Flesh of the Fuji Apple

  • Lim, Jeong-Ho;Jeong, Moon-Cheol;Moon, Kwang-Deog
    • Food Science and Biotechnology
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    • v.15 no.2
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    • pp.177-182
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    • 2006
  • Polyphenol oxidase (PPO) was isolated from the flesh of Fuji apples by DEAE-Cellulose, ammonium sulfate precipitation, phenyl-Sepharose CL-4B, and Sephdex G-100 chromatography. The molecular mass of the purified PPO was estimated to be 40 kDa by SDS polyacrylamide gel electrophoresis. With regard to substrate specificity, maximum activity was achieved with chlorogenic acid as substrate, followed by catechin and catechol whereas, there was no detectable activity with hydroquinic acid, resorcinol, or tyrosine as substrate. The optimum pH and temperature with catechol as substrate were 6.5 and $35^{\circ}C$, respectively. The enzyme was most stable at pH 6.0 and unstable at acidic pH. The enzyme was stable when it was heated to $45^{\circ}C$ but heating at $50^{\circ}C$ for more than 30 min caused 50% loss of activity. Reduced $ZnSO_4$, L-cystein, epigallocatechin-3-o-gallate (EGCG), and gallocatechin gallate (GCG) also inhibited activity.

Environmentally benign. Background-free protein staining in SDS-polyacrylamide gels using an counter ion-dye complex solution.

  • Jin , Li-Tai;Hwang, Sun-Young;Yoo, Gyurng-Soo;Choi, Jung-Kap
    • Proceedings of the PSK Conference
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    • 2002.10a
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    • pp.313.2-313.2
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    • 2002
  • Environmentally benign protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using both an acidic dye, zincon (ZC) and a basic dye. ethyl violet (EV) is described. It is based on a counter ion-dye staining technique that employs oppositely charged two dyes to form an ion-pair complex. The selective binding of the free dye molecules to proteins in an acidic solution produces bluish violet colored bands. It is a rapid procedure, involving only fixin and staining steps that are completed in 45 min. (omitted)

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Comparision of Mineral, Hydroxy Methyl Furfural Content and SDS-PAGE Pattern of Proteins in Different Honeys (다양한 꿀에 함유된 무기물 조성, Hydroxy Methyl Furfural 함량 및 꿀 단백질의 전기영동 패턴 비교)

  • Jung, Mi-Ea;Kim, Cheon-Jei;Paik, Hyun-Dong;Oh, Jae-Wook;Lee, Si-Kyung
    • Food Science of Animal Resources
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    • v.31 no.2
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    • pp.241-249
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    • 2011
  • This study was conducted to analyze ash content, mineral composition, hydroxy methyl furfural (HMF) content, stable carbon isotope ratio, and SDS-polyacrylamide gel electrophoresis patterns to investigate the quality characteristics of various honeys harvested from different sources and to identify differences useful for distinguishing honey sources. Ash content was 0.046-0.012% in acacia honey, 0.565-1.318% in chestnut honey, 0.06-0.582% in polyfloral honey, and 0.237-0.893% in native bee honey. Potassium content was high in order of chestnut honey>native bee honey>polyfloral honey>acacia honey. The Na/K ratio was 0.92-1.97 in acacia honey, 0.02-1.59 in chestnut honey, 0.02-5.30 in polyfloral honey, and 0.22-0.51 in native bee honey. The HMF content was 9.60-12.85, 10.15-25.75, 9.7-33.5, and 6.25-21.5 mg/kg in acacia, chestnut, native bee, and polyfloral honeys, respectively. HMF content was the highest in native bee honey. A 59 kDa protein band was revealed in all samples by SDS-PAGE analysis. Protein bands of 32.1, 31.9, and 33.5 kDa were revealed in some chestnut honeys, and protein bands of 32.3 and 32.5 kDa were shown in native bee honeys. A protein band of 72 kDa was also confirmed in some chestnut honeys.

Characteristics of Light Harvesting Chlorophyll-Protein Complex and Singlet Oxygen ($^1O_2$) Quenching in Leaf-burning Disease from Panax ginseng C. A. Meyer (인삼 Light Harvesting Chlorophyll Protein의 특성 및 엽소병에서 Singlet Oxygen($^1O_2$) Quenching)

  • 양덕조;이성택
    • Journal of Ginseng Research
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    • v.13 no.2
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    • pp.158-164
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    • 1989
  • In order to determine the relationships between the lea(-burning disease and the light harvesting chlorophyll-protein (LHCP) complex in Panax ginseng C. A. Meyer, we investigated the chlorophyll-protein (CP) complex of the thylakoid membrane and its characteristics. In P. ginseng four Cp-complex bands determined by non-denaturing SDS-PAGE were identified CP I'(containing reaction center of photosystem I and LHCP I antennae), CP I (reaction center of photosystem I) LHCP II** (oligoform of LHCP II), and LHCP II (photosystem II antennae, CP 26 and CP 29) by Bassis and Dunahay's procedures. Under our experimental condition, the CP I band was only observed in P. ginseng and the band intensity of LHCP II** in P ginseng was higher than in spinach and soybean. There were differences in the absorption and fluorescence spectra and chlorophyll a/b ratio of the CP-complex bands between P. ginseng and other Plants. The Polypeptidr content of P. ginseng thylakoid was lower than in spinach and soybean thylakoid, and the Polypeptide profiles of P. ginseng was low band intensity, especially about 29-35 kD, 55 kD, and 60 kD, compared to spinach and soybean. The inhibitory effects of 2,5-dimethylfuran, specific singlet oxygen ($^1O_2$) quencher, showed that singlet oxygen destroyed 60% of chl.a, 90% of chl.b and 70% of carotenoid in bleaching P. ginseng with leaf-burning disease.

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Characterization of Bacillus thuringiensis subsp. tohokuensis CAB167 Isolate against Mosquito Larva (모기유충에 활성 있는 Bacillus thuringiensis subsp. tohokuensis CAB167 균주의 특성)

  • Kil, Mi-Ra;Kim, Da-A;Paek, Seung-Kyoung;Kim, Jin-Su;Choi, Su-Yeon;Jin, Da-Yong;Youn, Young-Nam;Hwang, In-Chon;Ohba, Michio;Yu, Yong-Man
    • Korean journal of applied entomology
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    • v.47 no.4
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    • pp.457-465
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    • 2008
  • Eight Bacillus thuringiensis strains activated against mosquito larva were compared their characterization. Spherical-shaped parasporal inclusion of B. thuringiensis subsp. tohokuensis CAB167 was observed by phase-contrast microscopy and scanning electron microscopy. $LC_{50}$ values of B. thuringiensis subsp. tohokuensis CAB167 against Culex pipiens molestus, Culex pipiens pallens, and Aedes aegyti were 173, 190 and 580 ng/ml, respectively. B. thuringiensis subsp. tohokuensis CAB167 had a parasporal inclusion containing 4 major protein components, for example, 135, 80, 49 and 28-kDa by SDS-PAGE. Otherwise, after trypsin digestion of parasporal inclusion, SDS-PAGE was showed new protease-resistant peptides at 72 and 63-kDa. Activated toxins of isolated CAB167 were different from other reference strains on a serological by immuno-diffusion test.

The Effects of Cooking Temperature and Time on Gel Propertof Surimi-like Material from Porcine semimembranosus Musclye (돼지 반막양근을 이용한 수리미 유사물질의 겔 특성에 미치는 가열시간과 온도의 영향)

  • Han, Chul-Yong
    • Culinary science and hospitality research
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    • v.15 no.4
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    • pp.99-114
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    • 2009
  • This study examined the optimal cooking condition for surimi-like material (SLM) derived from porcine semimembranosus (SM) muscle and the effects of the various cooking temperatures and the cooking time on the gel properties. The most noticeable change that occurred during the preparation of the SLM from the SM was the reduction in the fat content (about 1%) during the washing procedures. The hardness and gel strength value were increased significantly as the cooking temperature was increased by $75^{\circ}C$ for 20 min (p<0.05). The SLMG cooked above $75^{\circ}C$ had a significantly higher WHC than the SLMG cooked below $75^{\circ}C$ (p<0.05). The gelling property of SLMG was effected for different conditions of cooking time and temperatures by the result of SDS-PAGE. After 20 min cooking, some enzyme bands including phosphorylase disappeared. The loss of these bands (about 46 kDa and 60 kDa) was observed after 20 min of cooking time. The photographs of microscopy showed that the filaments of myofibrils did not disappear after a cooking time of 15 min, and that the gaps between the fibers or filament were close. A significant change in the fibers and filaments occurred from 30 min to 35 min of cooking time, and the gradual coagulation of the structure of the SLM was observed with cooking time increased. These results suggest that a desirable surimi gel could be obtained from pork by cooking at $75^{\circ}C$ for 25 min.

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Effects of Several Medicinal Plants Extract on Survival Rate, Chlorophyll Contents and Photosynthetic Electron Transport Activity of Liverwort Photoautotrophic Cultured Cell (약용식물 추출액이 우산이끼 자가관양배양세포의 생존율, 엽록소함량 및 광합성전자전달 활성에 미치는 영향)

  • 정형진;권순태;김시무
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.40 no.2
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    • pp.133-141
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    • 1995
  • The effects of allelochemicals from medicinal plants have been studied as photo-synthetic inhibitor for photoautotrophic(PA) cultured cells. The extracts from 9 plant species were used for measuring the physiological effects on the liverwort cultured cell in following areas; germination inhibition, chlorophyll contents, hill activity, cell viability, photosynthetic oxygen evolution,and protein pattern changes on SDS PAGE. Germination inhibitions were detected in all plant after treating with 10% extract. Especially, treatment with 10% extract from Pulsatilla koreana and Aconitum carmichael inhibited germinations completely. Chlorophyll fornation was inhibited completely by treating PA cells with extract of Pulsatilla koreana, whose effect was similar to that of DCMU 10-3M, inhibitor for photosynthetic electron trans-fer. The treatment with extract from Pulsatilla koreana on PA cell showed the highest hill activity and the lowest cell viability among extracts studied. Oxygen releasing has been decreased down to 14-77% after treating with extracts from Pinellia ternata, Araliacont inentaila, Pulsatilla koreana and Vitex rotundifolia. Especially, 60$\mu$l of Pulsatilla koreana extract into 2ml mixture of PA cell inhibit-ed oxygen release up to 50%. Protein bands on SDS-PAGE, 14kD, 31kD, 41kD, 53kD, and 73kD, were not detected after treating Pulsatilla koreana extract on PA cells.

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