• Title/Summary/Keyword: $\beta$-Amino acid

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Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom -Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom-

  • Kim, Joung-Yoon;Lee, Seung-Bae;Kwon, Ki Rok;Choi, Suk-Ho
    • Journal of Pharmacopuncture
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    • v.17 no.1
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    • pp.44-50
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    • 2014
  • Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

한국산 장뇌산삼의 부위별 유용성분함량 및 추출용매조건의 영향

  • 김준한;문혜경;강우원;김종국
    • Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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    • 2003.10a
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    • pp.150.2-151
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    • 2003
  • 한국산 장뇌산삼의 열매, 잎, 줄기 및 뿌리를 -7$0^{\circ}C$ 동결건조 분말화시킨 시료에 대한 추출용매와 농도에 따른 유용성분의 함량을 비교, 분석하였다. 추출액의 당도는 잎과 줄기의 80% 에탄올추출액이 각각 22.58%와 22.53%로 가장 높았고, pH는 4.43-7.41 수준이었고, 갈변도는 흡광도가 잎과 뿌리 80% 에탄올추출액이 1.803과 1.085로 가장 높았다. 색도의 경우 L값는 줄기 100% 증류수추출액이 24.56, 열매 80% 메탄올추출액이 24.35로 가장 높았고, 뿌리와 잎 80% 에탄올추출액이 각각 17.47과 19.59로 가장 낮은 값을 나타내었다. a값은 잎100% 증류수추출액이 0.41로 가장 높았고, 줄기 80%메탄올추출 액이 -0.49로 가장 낮은 값을 보였다. b값은 줄기 80% 메탄올추출액이 3.69로 가장 높았고, 열매 100% 증류수추출액 이 0.45로 가장 낮은 값이었다. 주요 유리당은 sucrose, glucose 및 fructose 이었고, 유리당 총함량은 열매와 줄기 100% 증류수추출액이 각각 6733 mg/100g과 6142 mg/100g으로 가장 많은 량을 함유하였고, sucrose는 뿌리 80% 메탄올추출액 이 3673 mg/100g으로, glucose 및 fructose는 줄기 80% 에탄올추출액과 잎 80% 메탄올추출액에 각각 4283 mg/100g과 1897 mg/100g으로 높은 함유량을 나타내었다. 또한 잎과 줄기에는 xylose가 304-524 mg/100g 수준으로 함유되어 있었고, 뿌리에는 maltose가 소량 함유하고 있었다. 주요 유기산으로는 citric acid, tartaric acid 및 malic acid가 확인되었고, citric acid는 뿌리 80% 메탄올추출액이 1849 mg/100g으로 가장 높은 함량이었고, tartaric acid는 잎 80% 메탄올추출액이 3263 mg/100g으로 가장 높은 함량이었고, malic acid는 뿌리 80% 메탄올추출액이 1856 mg/100g으로 가장 높은 함량이었으며, 뿌리 80% 메탄올추출액에는 succinic acid, malonic acid 및 oxalic acid 등이 확인되었다. 유리아미노산은 L-Arginine, ${\gamma}$-Amino-n-butyric acid, Ethanolamine, L-Proline, $\beta$-Alanine 및 L-sarcosine 등 총 35종이 확인되었고, L-Arginine은 251-7379 mg/100g 수준으로 특히, 뿌리 80% 메탄올추출액에는 1319 mg/100g으로 총함유량의 79.13%로 매우 높은 함유량을 나타내었다. P, K, Na, Ca 및 Mg 등이 주된 무기질로 확인되었고, 그 중 P는 줄기 100% 증류수추출액에 15563 mg/100g으로 가장 높았고, K은 잎 80% 메탄을 추출액에 4952 mg/100g, Ca과 Na은 잎과 열매 100% 증류수추출액에 각각 3052 mg/100g과 1798 mg/100g, Mg은 잎 100% 증류수추출액에 950 mg/100g으로 매우 높은 함유량을 보였고, 또한 미량원소로는 Zn, Cu, Cr, Mn, Co, Mo, Fe 등이 함유되어 있었다.

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Nutritional Components of Korean Traditional Actinidia (Actinidia arguta) Sprout and in vitro Antioxidant Effect (한국 토종다래(Actinidia arguta)순의 주요 영양성분 및 in vitro 항산화 활성)

  • Jin, Dong Eun;Park, Seon Kyeong;Park, Chang Hyeon;Seung, Tae Wan;Choi, Sung-Gil;Heo, Ho Jin
    • Korean Journal of Food Science and Technology
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    • v.47 no.1
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    • pp.37-43
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    • 2015
  • Nutritional composition and in vitro antioxidant activities of the ethanol extract of Korean traditional actinidia (Actinidia arguta) sprouts of the Otumsense variety were investigated to examine the sproutsi nutritional value. The most abundant mineral, amino acid, and fatty acid were calcium, glutamic acid, and ${\alpha}$-linolenic acid, respectively. The major free sugar of Otumsense sprouts was sucrose. The level of vitamin C, a natural antioxidant, was highest among other vitamins examined. The amount of total polyphenol was highest in the 40% ethanol extract. The 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) radical scavenging activity of the 40% ethanol extract was about 94% at a concentration of $1,000{\mu}g/mL$. Malondialdehyde inhibition by the extract increased in a dose-dependent manner (from 0 to $100{\mu}g/mL$). Intracellular reactive oxygen species accumulation resulting from $H_2O_2$ treatment of PC12 cells significantly reduced when the 40% ethanol extract was present in the media compared to that in PC12 cells treated with $H_2O_2$ only.

Cloning, Expression, and Characterization of Protease-resistant Xylanase from Streptomyces fradiae var. k11

  • Li, Ning;Yang, Peilong;Wang, Yaru;Luo, Huiying;Meng, Kun;Wu, Nigfeng;Fan, Yunliu;Yao, Bin
    • Journal of Microbiology and Biotechnology
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    • v.18 no.3
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    • pp.410-416
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    • 2008
  • The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

A Novel Glycosyl Hydrolase Family 16 β-Agarase from the Agar-Utilizing Marine Bacterium Gilvimarinus agarilyticus JEA5: the First Molecular and Biochemical Characterization of Agarase in Genus Gilvimarinus

  • Lee, Youngdeuk;Jo, Eunyoung;Lee, Yeon-Ju;Hettiarachchi, Sachithra Amarin;Park, Gun-Hoo;Lee, Su-Jin;Heo, Soo-Jin;Kang, Do-Hyung;Oh, Chulhong
    • Journal of Microbiology and Biotechnology
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    • v.28 no.5
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    • pp.776-783
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    • 2018
  • The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

Monthly Variations in the Nutritional Composition of Antarctic Krill Euphausia superba

  • Kim, Min-A;Jung, Hae-Rim;Lee, Yang-Bong;Chun, Byung-Soo;Kim, Seon-Bong
    • Fisheries and Aquatic Sciences
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    • v.17 no.4
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    • pp.409-419
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    • 2014
  • The proximate composition and various specific components of Antarctic krill Euphausia superba, in the catch season between March and August were investigated. Frozen krill were freeze-dried and milled. The proximate composition comprised water, proteins, fats, ash, fatty acids, and amino acids, while the specific components were vitamins, minerals, nucleotides, betaine, and astaxanthin. The moisture content of the krill ranged from 77 to 80%, with the highest value in June, and the ash content was between 12 and 13%. The protein content was lowest in May, and the fat content was 18-19%, with the highest value in March. The amino acid content varied according to the season: taurine and glycine were highest in August; ${\beta}$-alanine was higher in April and May; and arginine, ornithine, and lysine were highest in March. The unsaturated fat content was ~50% and omega-3 fatty acids were highest in June. Oil-soluble vitamins A and E were highest in March, and the water-soluble vitamin content was less than that of oil-soluble vitamins. The mineral content was highest in June, and the most abundant mineral was sodium at 235.60 mg/100 g krill. The content of other minerals was lowest (2.94 mg/100 g) in April, except for lead. The nucleotide content was highest in July, while the betaine content was highest in April and lowest in June. The astaxanthin content was highest in May and ranged from 6 to 10 ppm in other months.

OPTHiS Identifies the Molecular Basis of the Direct Interaction between CSL and SMRT Corepressor

  • Kim, Gwang Sik;Park, Hee-Sae;Lee, Young Chul
    • Molecules and Cells
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    • v.41 no.9
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    • pp.842-852
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    • 2018
  • Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to ${\beta}-trefoil$ domain (BTD) of CSL using a conserved ${\varphi}W{\varphi}P$ motif (${\varphi}$ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ${\varphi}W{\varphi}P$ sequence (APIWRP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.

An ${\beta}$-1,4-Xylanase with Exo-Enzyme Activity Produced by Paenibacillus xylanilyticus KJ-03 and Its Cloning and Characterization

  • Park, Dong-Ju;Lee, Yong-Suk;Chang, Jie;Fang, Shu-Jun;Choi, Yong-Lark
    • Journal of Microbiology and Biotechnology
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    • v.23 no.3
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    • pp.397-404
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    • 2013
  • Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

Analysis of Structure and Expression of Grapevine 2-oxoglutarate Oxygenase Genes in Response to Low Temperature

  • Kim, Seon Ae;Ahn, Soon Young;Yun, Hae Keun
    • Horticultural Science & Technology
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    • v.34 no.1
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    • pp.46-54
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    • 2016
  • 2-Oxoglutarate (2OG) acts as a signaling molecule and plays a critical role in secondary metabolism in a variety of organisms, including plants. Six 2-oxoglutarate (2OG) and Fe(II) oxygenase (2OGO) genes, VlCE2OGO1 [Vitis labruscana 2-oxoglutarate (2OG) and Fe(II) oxygenase 1], VlCE2OGO2, VlCE2OGO3, VlCE2OGO4, VlCE2OGO5, and VlCE2OGO6, which show different expression patterns upon transcriptome analysis of 'Campbell Early' grapevine exposed to low temperature for 4 weeks, were analyzed for their structure and expression. Comparison of the deduced amino acid sequences of the 2OGO genes from the V. labruscana transcripts revealed sequence similarities of 38.6% (VlCE2OGO1 and VlCE2OGO2) to 19.2% (VlCE2OGO2 and VlCE2OGO3). The lengths of these genes ranged from 1053 to 2298 bp, and they encoded 316 to 380 amino acids. The prediction of the secondary structure of the encoded proteins by Self-Optimized Prediction Method with Alignment (SOPMA) indicated that all the genes contained alpha helix (23.95 to 41.71%), extended strand (16 to 22.34%), beta turn (6.65 to 9.22%), and random coil (32.97 to 51.58%) in the analysis. Specific primers from unique regions in each gene obtained by alignment of nucleotide sequences were used in real time PCR for analysis of gene expression. All tested genes showed differential expression in grapevines exposed to low temperature. Of the six transcripts, VlCE2OGO1, VlCE2OGO2, and VlCE2OGO3 were up-regulated and VlCE2OGO4, VlCE2OGO5, and VlCE2OGO6 were down-regulated in response to cold treatments at all tested time points. The 2OG genes can be used for elucidation of mechanisms of tolerance to cold and as valuable molecular genetic resources for selection in breeding programs for cold-hardy grapevines.

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

  • You, Eunae;Jeong, Jangho;Lee, Jieun;Keum, Seula;Hwang, Ye Eun;Choi, Jee-Hye;Rhee, Sangmyung
    • BMB Reports
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    • v.55 no.4
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    • pp.192-197
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    • 2022
  • Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, significantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.