• Title/Summary/Keyword: Glycine site

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Effect of Mutagenesis of V111 and L112 on the Substrate Specificity of Zymomonas mobilis Pyruvate Decarboxylase

  • Huang, Chang-Yi;Nixon, Peter F.;Duggleby, Ronald G.
    • BMB Reports
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    • v.32 no.1
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    • pp.39-44
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    • 1999
  • Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

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Content Differences of Pb and Cd by Trophic Levels of the Ecosystem near Highways with Different Traffic Volumes (교통량이 다른 고속도로변 생태계의 영양단계별 납과 카드뮴 함량의 차이)

  • Chung, Wan-Ho;Hai-Poong Lee
    • The Korean Journal of Ecology
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    • v.10 no.1
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    • pp.7-16
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    • 1987
  • The content of Pb and Cd in the soil, producer, 1st consumer and 2nd consumer by the distance from two highway with different construction years and traffic volumes was measured from 1984 through 1986. The result was compared with that of a control in Yangsu-ri near Mt. Unak, Namyangju-gun, Kyunggi-do. The producers in the area of Yangjae-done are composed of 27 species of floras with Glycine soja being the dominant species while those in Shingal 20 species, with Leesia japonica being the dominant species. The faunas in the former area include 37 families and 69 species, with insects being dominant. In the area of Yangjae-dong, the biomass by trophic levels increased from the ration of 1/400 between producer and 1st consumer to the ratio of 1/10 between 1st consumer and 2nd consumer. The Pb content of soil, producer, 1st consumer and 2nd consumer in Yangjae-done near the Kyungbu highway with traffic jam has the sigingacant difference of 1% level at the vicinity of 10 meters and it is the same with that of Shingal area nearthe Youngdong highway with relatively sparse traffic except for the 2nd consumer. In case of cadmium content, the 1st and 2nd consumer has 1% signigicant difference near the Kyungbu highway and other trophic levels have no diference. Besides, the content of Pb and Cd at the site 1, 2 and 3 within the range of 100 meters from the two highways has 1% significant difference from that of a control while the site 4 has no difference.

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Metarizium anisopliae (Metschn.) Sorok이 생산하는 Lactobacillus plantarum 용균효소의 분리, 정제 및 특성

  • Ryoo, Ky-Chul;Hahm, Byoung-Kwon;Paik, Un-Wha;Yu, Ju-Hyun;Bai, Dong-Hoon
    • Microbiology and Biotechnology Letters
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    • v.24 no.6
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    • pp.678-686
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    • 1996
  • To improve the preservation of Kimchi, we isolated Lactobacillus plantarum lytic enzyme-producing strain from soil, and the enzyme was purified and characterized. From the observation of cultural and morpho- logical characteristics, the isolated strain was identified as Metarrisium anisopliae (Metschn.) Sorok. The enzyme was purified to 75-folds with 40% yields through affinity adsorption and CM-Sephadex C-50 column chromatog- raphy. The optimum pH and temperature for lytic activity are 4.0 and 40$\circ$C, respectively, and the enzyme acitvity is stable between pH 3.0 and 9.0, and up to 50$\circ$C. The enzyme is a monomeric protein with molecular weight of 40,000 daltons by SDS-PAGE and gel filtration. The enzyme is endopeptidase which breaks the peptide linkage of Lactobacillus plantarum peptidoglycan. The lytic action spectra confirmed that Leuconostoc mesenteroides, a useful strain for the fermentation of Kimchi, is not lysed by the enzyme. The enzyme activity is inhibited by N-bromosuccinimide (NBS), which probably indicates the involvement of tryptophan residue in active site of the enzyme, and also inhibited by Ag$^{+}$. The amino acid composition analysis showed that the enzyme contains more acidic amino acids than basic ones, and composition of alanine, glycine, proline and tyrosines was very high.

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The Study of Cyclophosphamide Metabolite $^{15}N$ and $^{17}O$ Phosphoramide Mustards (항암제인 Cyclophosphamide의 중간체인 $^{15}N$$^{17}O$-phosphoramide Mustards의 합성)

  • Koo, Kyo-Im;Ryem, Kon
    • YAKHAK HOEJI
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    • v.38 no.4
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    • pp.455-461
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    • 1994
  • Each nitrogen and oxygen site isotope enriched the cyclophosphamide metabolite phosphoramide mustard was synthesized. Reaction of N,N-bis(2-chloroethyl)phosphoramidic dichloride$[Cl_2P(O)N(CH_2CH_2Cl)_2]$ with benzyl alcohol and ammonia gave N,N-bis(2-chloroethyl)phosphorodiamidic acid phenylmethyl ester $[BzO(H_2N)P(O)N(CH_2CH_2Cl)_2]$. Catalytic hydrogenation of this benzyl ester followed by the addition of cyclohexylamine provided PM. Incorporation of $^{15}NH_3$ into this general scheme gave PM with a $^{15}NH_2$ moiety. Glycine-$^{15}N$ was converted to bis(2-chloroethyl)amine-$^{15}N$ hydrochloride which, in turn, provided for N,N-bis(2-chloroethyl)phosphorodiamidic-$^{15}N$ dichloride. Use of this compound in the general synthetic pathway yielded PM CHA with $^{15}N$ in the mustard moiety. $^{17}O$-Enriched PM was generated through the use of benzyl alcohol-$^{17}O$. To obtain the alcohol, labelled benzaldehyde was made by exchange with $^{17}OH_2$ and was then reduced with sodium borohydride.

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Gene Duplications Revealed during the Process of SNP Discovery in Soybean[Glycine max(L.) Merr.]

  • Cai, Chun Mei;Van, Kyu-Jung;Lee, Suk-Ha
    • Journal of Crop Science and Biotechnology
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    • v.10 no.4
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    • pp.237-242
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    • 2007
  • Genome duplication(i.e. polyploidy) is a common phenomenon in the evolution of plants. The objective of this study was to achieve a comprehensive understanding of genome duplication for SNP discovery by Thymine/Adenine(TA) cloning for confirmation. Primer pairs were designed from 793 EST contigs expressed in the roots of a supernodulating soybean mutant and screened between 'Pureunkong' and 'Jinpumkong 2' by direct sequencing. Almost 27% of the primer sets were failed to obtain sequence data due to multiple bands on agarose gel or poor quality sequence data from a single band. TA cloning was able to identify duplicate genes and the paralogous sequences were coincident with the nonspecific peaks in direct sequencing. Our study confirmed that heterogeneous products by the co-amplification of a gene family member were the main cause of obtaining multiple bands or poor quality sequence data in direct sequencing. Counts of amplified bands on agarose gel and peaks of sequencing trace suggested that almost 27% of nonrepetitive soybean sequences were present in as many as four copies with an average of 2.33 duplications per segment. Copy numbers would be underestimated because of the presence of long intron between primer binding sites or mutation on priming site. Also, the copy numbers were not accurately estimated due to deletion or tandem duplication in the entire soybean genome.

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A Novel Glycine-Rich Region in Sox4 is a Target for the Proteolytic Cleavage in E. coli (전사활성 인자인 Sox4의 단백질 분해효소에 의한 표적 부위에 관한 연구)

  • 허은혜;최주연;장경희;김인경;임향숙
    • Korean Journal of Microbiology
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    • v.38 no.3
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    • pp.153-161
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    • 2002
  • Sox4, a transcription factor, consists of three functional domains: an HMG-box domain as a DNA binding domain, serine rich region as a transactivation domain and glycine rich region (GRR), an unknown functional domain. Although Sox4 is known to be functionally involved in heart, B-cell and reproductive system development, its physiological function remains to be elucidated. We used pGEX expression system to develop a simple and rapid method for purifying Sox4 protein in suitable forms for biochemical studies of their functions. Unexpectedly, we observed that full-length Sox4 appears to be protease-sensitive during expression and purification in E. coli. To map the protease-sensitive site in Sox4, we generated various constructs with each of functional domains of Sox4 and purified as the GST-Sox4 fusion proteins using glutathione beads. We found that the specific cleavage site for the proteolytic enzyme, which exists in E. coli, is localized within the novel GRR of Sox4. Our study suggest that the GRR of Sox4 may a target for the cellular protease action and this cleavage in the GRR may be involved in regulating physiological function of Sox4. Additionally, our study may provide a useful method for investigating the proteolytic cleavage of the target molecule in E. coli.

Transport System of Specific Neutral Amino Acids in Suspension-Cultured Cells (현탁배양 세포내에서 특수 중성 아미노산의 수송)

  • Bong-Heuy CHO
    • Korean Journal of Plant Tissue Culture
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    • v.21 no.4
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    • pp.201-206
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    • 1994
  • The influx of glycine, valine, alanine, and histidine was inhibited by all tested neutral amino acids competitively and the reciprocal inhibitory studies showed the neutral amino acids possess the same transport system as neutral amino acids process to the same catalytic site of one carrier to each other, The molecules of histidine were transported actively as a neutral form through the neutral amino acid transport system but were not transported as a charged form. The Km values of the neutral amino acid transport system have been divided into three different category on basis of the affinity to the carrier, below 0.1mM, etween 0.1ImM-0.5mM and above 0.5mM. The $V_{max}$ was between $3.12{\mu}mole{\cdot}h^{-1}{\cdot}g$ fresh $weight^{-1}\;-\;15.1\;{\mu}mole{\cdot}h^{-1}{\cdot}g$ fresh $weight^{-1}$. Neutral amino acids cotransported with one $H^{+}per$ one molecule and one $K^{+}-efflux$ per one molecule for charge compensation. Histidine cotransported with proton per one molecule, however the movement of cotransported proton can't detectable because of the release of proton from the charged molecules of histidine in the medium.

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Leaf Photosynthesis as Influenced by Mesophyll Cell Volume and Surface Area in Chamber-Grown Soybean (Glycine max) Leaves (중엽세포의 체적 및 표면적과 콩잎의 광합성 능력간 관계)

  • Jin Il, Yun;S. Elwynn, Taylor
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.33 no.4
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    • pp.353-359
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    • 1988
  • Variations in photosynthetic capacities of leaves differing in thickness were explained on the basis of relationships between gas exchange and internal leaf structure. The relative importance of gas diffusion and of biochemical processes as limiting for leaf photosynthesis was also determined. Mesophyll cell surface was considered to be the limiting internal site for gas diffusion. and cell volume to be indicative of the sink capacity for CO$_2$ fixation. Increases in cell surface area were assumed to reduce proportionately mesophyll resistance to the liquid phase diffusion of CO$_2$. Increased cell volume was thought to account for a proportional increase in reaction rates for carboxylation, oxygenation. and dark respiration. This assumption was tested using chamber-grown Glycine max (L.) Merr. cv. Amsoy plants. Plants were grown under 200, 400, and 600 ${\mu}$mol photons m$\^$-2/ s$\^$-1/ of PAR to induce development of various leaf thickness. Photosynthetic CO$_2$ uptake rates were measured on the 3rd and 4th trifoliolate leaves under 1000 ${\mu}$mol photons m$\^$-2/ s$\^$-1/ of PAR and at the air temperature of 28 C. A pseudo -mechanistic photosynthesis model was modified to accommodate the concept of cell surface area as well as both cell volume and surface area. Both versions were used to simulate leaf photosynthesis. Computations based on volume and surface area showed slightly better agreement with experimental data than did those based on the surface area only. This implies that any single factor, whether it is photosynthetic model utilized in this study was suitable for relating leaf thickness to leaf productivity.

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Effects of elevated-$CO_2$ concentration and -temperature on the phenological and reproductive responses of Baktae and Seomoktae, Glycine max (L.) Merrill ($CO_2$농도와 온도 상승에 따른 백태와 서목태(Glycine max (L.) Merrill)의 식물계절학 및 번식생태학적 반응)

  • Park, Jae-Hoon;Cho, Kyu-Tae;Kim, Sang-Bum;Jang, Rae-Ha;You, Young-Han
    • Korean Journal of Environment and Ecology
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    • v.28 no.6
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    • pp.634-641
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    • 2014
  • In order to elucidate the ecological and phenological responses of bean under global warming situation, we investigated leafing and flowering phenomena and growth traits of Baktae and Seomoktae in control plot (ambient $CO_2$ concentration and temperature) and warmed plot (elevated $CO_2$ concentration and increased temperature than control) in green house. Average $CO_2$ concentration and temperature were set 540 ppm in treatment and higher $2.2^{\circ}C$ in treatment than control. The appearance of leaf unfolding and the initiation of blooming and fruit maturing in Seomoktae were late in treatment than control. In case of Baktae, the number of total pods and seeds per plant in treatment was fewer than that in control. For Seomoktae, the number of total pods per plant and weight of total seeds per plant were decreased in treatment than in control. Thus, the number of pods and seeds of Baktae declined and the production of Seomoktae was decreased under global warming condition. This result indicates that site selection and crop yield for cultivating of the bean may be changed into new area due to global warming condition.

Seasonal variation in the body composition, amino acid, fatty acid and glycogen contents of triploid Pacific oyster, Crassostrea gigas in western coastal waters of Korea (서해안 3배체 굴 (Crassostrea gigas) 의 계절별 체성분, 아미노산, 지방산 및 글리코겐 함량 변화)

  • Hwang, In Joon;Han, Jong Chul;Hur, Young Baek;Lim, Hyun Jeong
    • The Korean Journal of Malacology
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    • v.32 no.4
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    • pp.269-277
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    • 2016
  • We examined the seasonal variation of the proximate, amino acid, fatty acid composition and glycogen contents of triploid Pacific oyster cultured in western coastal waters of Korea in 2015. The specimens were collected in February, May, August and November from off-bottom farming site. The lipid content was the highest in winter season (February, $2.10{\pm}0.06%$), the protein content was the highest in autumn season (November, $9.09{\pm}0.04%$) while the moisture and ash content was the highest in summer season (August, $86.00{\pm}0.12%$ and $2.67{\pm}0.03%$, respectively). The total amino acid contents was the highest in winter season (1,011.5 mg / 100 g), decreased gradually and was the lowest in summer season (486.7 mg / 100 g). The total amino acid composition was dominated by taurine, aspartic acid, glutamic acid, glycine and alanine. In the fatty acid composition, the two fatty acids, eicosapentaenoic acid and docosahexaenoic acid, which comprised that as much as 50% of the total fatty acid contents, were dominated during all season. The glycogen content was the highest in summer season ($1.26{\pm}0.03g$ / 100 g) although it was stable during the other season with the range of $0.69{\pm}0.04-0.86{\pm}0.01g$ / 100 g. Taken together, these results suggest that triploid pacific oysters have high commercial value not only in winter season but in summer season because they do not spawn and have high contents of glycogen comparing with those of diploid oyster in this period.