• Title/Summary/Keyword: Covalently

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Protein unfolding by ATP-dependent proteases

  • Lee, Cheolju;Michael Schwartz;Sumit Prakash;Masahiro Iwakura;Andreas Matouschek
    • Proceedings of the Korean Biophysical Society Conference
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    • 2003.06a
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    • pp.34-34
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    • 2003
  • Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. C1pAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP, These proteases catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. An ${\alpha}$-helix is easier to unravel than a ${\beta}$-strand. In multidomain proteins, independently stable domains are unfolded sequentially. The steric constraints imposed on substrate proteins during their degradation by the proteasome were investigated by constructing a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. Our results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain, and also explain the limited degradation by the proteasome that occurs in the processing of the transcription factor NF-KB, and also implicate difficulty in degradation of amyloidal aggregates by the proteasome

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Sensing Characteristics of Tyrosinase Immobilized and Tyrosinase, Laccase Co-immobilized Platinum Electrodes

  • Quan, De;Kim, You-Sung;Shin, Woon-Sup
    • Bulletin of the Korean Chemical Society
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    • v.25 no.8
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    • pp.1195-1201
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    • 2004
  • Tyrosinase was covalently immobilized on platinum electrode according to the method we developed for laccase (Bull. Korean Chem. Soc. 2002, 23(7), 385) and p-chlorophenol, p-cresol, and phenol could be detected with sensitivities of 334, 139 and 122 nA/ ${\mu}M$ and the detection limits of 1.0, 2.0, and 2.5 ${\mu}M$, respectively. The response time ($t_{90\%}$) is 3 seconds for p-chlorophenol, and 5 seconds for p-cresol and phenol. The optimal pHs of the sensor are in the range of 5.0- 6.0. This sensor can tolerate at least 500 times repeated injections of p-chlorophenol with retaining 80% of initial activity. In case of tyrosinase and laccase co immobilized platinum electrode, the sensitivities are 560 nA/ ${\mu}M$ for p-phenylenediamine (PPD) and 195 nA/ ${\mu}M$ for p-chlorophenol, respectively. The sensitivity of the bi-enzyme sensor for PPD increases 70% compared to that of only laccase immobilized one, but the sensitivity for p-chlorophenol decreases 40% compared to that of only tyrosinase immobilized one. The sensitivity increase for the bi-enzyme sensor for PPD can be ascribed to the additional catalytic function of the co-immobilized tyrosinase. The sensitivity decrease for p-chlorophenol can be explained by the “blocking effect” of the co-immobilized laccase, which hinders the mass transport through the immobilized layer. If PPD was detected with the electrode that had been used for p-chlorophenol, the sensitivity decreased 20% compared to that of the electrode that had been used only for PPD. Similarly, if p-chlorophenol was detected with PPD detected electrode, the sensitivity also decreased 20%. The substrate-induced conformation changes of the enzymes in a confined layer may be responsible for the phenomena.

Softening Related Changes in Cell Wall Pectin of Hot Pepper Fruit (고추과실 세포벽 Pectin질의 연화에 따른 변화)

  • Kim, Soon-Dong;Yoon, Soo-Hong;Kang, Meung-Su;Park, Nam-Sook
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.15 no.2
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    • pp.171-175
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    • 1986
  • Pectic substance of hot pepper crude cell wall was fractionated during ripening and softening. The pectic substance was divided ionically associated pectin(IAP) and covalently bounded pectin with hemicellulose(CBP). And then, the composition and the modification of the pectic substance using gel filtration chromatography were studied. The polyuronide associated with the CBP increased owing to decreasing of pectin bound hemicellulose linked to galactose by the turning stage showed softening. The molecular weight profiles of IAP shifted from low molecular weight profiles of IAP shifted from low molecular weight to high molecular weight polymers. While, the CBP changed from high molecular weight to low molecular weight polymers during ripening. It is suggested that the changes in the pectic substance associated with fruit softening should involve the alteration of CBP-bound hemicellulose like galactan.

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Research of Characterization of Covalently Cross-linked SPEEK/Cs-Substituted MoPA/Ceria 1wt% Composite Membrane for Water Electrolysis (Cs 치환에 따른 수전해용 공유가교 SPEEK/Cs(n)-MoPA/Ceria 1%(n = 1~3) 복합막의 특성 연구)

  • Park, Daeyoung;Hwang, Sungha;Oh, Seunghee;Yoon, Daejin;Kang, Ansoo;Moon, Sangbong;Chung, Janghoon
    • Transactions of the Korean hydrogen and new energy society
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    • v.25 no.1
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    • pp.1-10
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    • 2014
  • Ceria ($CeO_2$) was used to scavenge free radicals which attack the membrane in the polymer electrolyte membrane water electrolysis (PEMWE) circumstance and to increase the duration of the membrane. In order to improve the electrochemical, mechanical and electrocatalytic characteristics, engineering plastic of the sulfonated polyether ether ketone (SPEEK) as polymer matrix was prepared in the sulfonation reaction of polyether ether ketone (PEEK) and the organic-inorganic blended composite membranes were prepared by sol-gel casting method with loading the highly dispersed ceria and cesium-substituted phophomolybdic acid(Cs-MoPA) with cross-linking agent contents of 0.01mL. In conclusion, CL-SPEEK/$Cs_{(2.5)}$-MoPA/ceria(1%) membrane showed the optimum results such as 0.1095S/cm of proton conductivity at $80^{\circ}C$, 2.906meq./g-dry-membrane of ion exchange capacity and mechanical characteristics, and 49.73MPa of tensile strength which were better than Nafion 117 membrane.

Enzymatic Synthesis of Meth.yl Fructoside by Immobilized Invertase (고정화 전화당 효소에 의한 메틸 프룩토시드의 합성)

  • 허주형;김해성
    • KSBB Journal
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    • v.8 no.4
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    • pp.313-319
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    • 1993
  • Methyl fructoside was synthesized from sucrose and methanol using an immobilized invertase. The enzyme was covalently bound by glutaraldehyde on porous silica coated with polyethyleneimine to give loading capacity of 120mg of invertase per one gram of dry porous silica and effective activity of 100U per one milligram of bound invertase. Polyethyleneimine coating imparted a hydrophillic character, good activity retention and high loading capacity to the surface of porous silica as well as hydrophillic microenviroment in the vicinity of bound invertase. The immobilized enzyme was formed into an alginate-enclosed silica bead to have enough activity for methyl fructoside synthesis from aqueous methanol-sucrose solution. Using the alginate-enclosed biocatalyst the yield of methyl fructoside was obtained as high as 55.9% from aqueous 30% (v/v) methanol and 0.291mo1/l sucrose with 2U/ml activity at $25^{\circ}C$, pH 4.8.

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Injectable TGF-beta 3-conjugated hyaluronic acid hydrogel for cartilage regeneration

  • Ko, Ki Seong;Lee, Jung Seok;Park, Kyung Min;Lee, Yunki;Oh, Dong Hwan;Son, Joo Young;Kwon, Oh Hee;Eom, Min Yong;Park, Ki Dong
    • Biomaterials and Biomechanics in Bioengineering
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    • v.2 no.1
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    • pp.23-32
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    • 2015
  • Facile immobilization of growth factors in hyaluronic acid (HA) hydrogels using dual enzymes is reported in the paper. The hydrogels were formed by using horseradish peroxidase (HRP) and hydrogen peroxide ($H_2O_2$) and transforming growth factor-${\beta}3$ (TGF-${\beta}3$) was covalently conjugated on the hydrogels in situ using tyrosinase (Ty) without any modifications. For the preparation of hydrogels, HA was grafted with poly(ethylene glycol) (PEG), which was modified with a tyrosine. The gelation times of the HA hydrogels were ranging from 415 to 17 s and the storage moduli was dependent on the concentration of $H_2O_2$ and Ty (470-1600 Pa). A native TGF-${\beta}3$ (200 ng/mL) was readily encapsulated in the HA hydrogels and 17% of the TGF-${\beta}3$ was released over 1 month at the Ty concentration of 0.5 KU/mL, while the release was faster when 0.3 KU/mL of Ty was used for the encapsulation (27%). It can be suggested that the growth factors resident in the hydrogels for a long period of time may lead cells proliferating and differentiating, whereas the growth factors that are initially released from the hydrogels can induce the ingrowth of cells into the matrices. Therefore, the dual enzymatic methods as facile gel forming and loading of various native growth factors or therapeutic proteins could be highly promising for tissue regenerative medicines.

A Study on Optical Properties of Nanocomposite Composed of Au Nanorods and Organic Dyes (금 나노막대와 유기 염료로 구성된 나노복합체의 광학특성 연구)

  • Kim, Ki-Se;Yoo, Seong Il;Sohn, Byeong-Hyeok
    • Clean Technology
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    • v.20 no.2
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    • pp.141-145
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    • 2014
  • In this study, we studied optical properties on the layer-by-layer (LbL) assemblies consisting of Au nanorods and organic dyes. For this purpose, poly (allylamine hydrochloride), PAH and poly (styrene sulfonate), PSS were selected as ionic polymers and rhodamine B isothiocyanate (RB) was utilized as an organic dye based on its spectral overlap with plasmon band of Au nanorods. In the view point of assembling methods, RB was covalently attached to PAH, then, LbL structure of Au [PSS/PAH]2/PSS/PAH-RB was prepared by sequential coating of PAH, PSS, PAH-RB on Au nanorods. Since the prepared LbL assembly exhibits both plasmonic and fluorescent properties, we studied the mutual nanorod-dye properties by dissolving Au nanorods.

Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

  • Bang, Injin;Choi, Hee-Jung
    • Molecules and Cells
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    • v.38 no.2
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    • pp.105-111
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    • 2015
  • The beta2-adrenergic receptor (${\beta}2AR$) belongs to the G protein coupled receptor (GPCR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound ${\beta}2AR$ in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, ${\beta}2AR$ is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of ${\beta}2AR$. In this review, structural features of inactive and active states of ${\beta}2AR$, the interaction of ${\beta}2AR$ with heterotrimeric G protein, and the comparison with ${\beta}1AR$ will be discussed.

Advances of Post-combustion Carbon Capture Technology by Dry Sorbent (건식흡수제 이용 연소배가스 이산화탄소 포집기술)

  • Yi, Chang-Keun
    • Korean Chemical Engineering Research
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    • v.48 no.2
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    • pp.140-146
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    • 2010
  • This paper addresses recent status and trends of carbon dioxide capture technologies using dry sorbents in the flue gas. The advantages of dry sorbent $CO_2$ capture technology are broader operating temperature range, less energy loss, less waste water, less corrosion problem, and natural properties of solid wastes. Recently, U.S.A. and Korea have been developing processes capturing $CO_2$ from real coal flue gas as well as sorbents improving sorption capacity to decrease total $CO_2$ capture cost. New class of dry sorbents have been developed such as chemisorbents with alkali metals of which material cost is low, amines physically adsorbed on silica supports, amines covalently tethered to the silica support, carbon-supported amines, polymer-supported amines, amine-containing solid organic resins and metal-organic framework. The breakthrough is needed in the materials on dry sorbents to decrease capture cost.

Electrochemical Studies of Immobilized Laccases on the Modified-Gold Electrodes

  • Yoon Chang-Jung;Kim Hyug-Han
    • Journal of the Korean Electrochemical Society
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    • v.7 no.1
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    • pp.26-31
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    • 2004
  • The direct electrochemical studies of four laccases (plant and fungal laccases) have been investigated on a gold electrode functionalized with a new tether of 2.2'-dithiosalicylic aldehyde. Results from these studies indicate that the redox potential of the active site of plant laccase from Rhus vernificera is shifted to a more negative value(255 mV versus SCE) than that of fungal laccase from Pyricularia oryzae (480 mV versus SCE). Mechanistic studies indicate that the reduction of type-1 Cu precedes the reduction of type-2 and type-3 Cu ions when the electrode is poised initially at different potentials. Also a new tether, 2.2'-dithiosalicylic aldehyde, has been used to study the redox properties of two laccases (LCCI and Lccla) covalently attached to a gold electrode. An irreversible peak at 0.47V vs. SCE is observed in the cyclic voltammorams of LCCI. In contrast, the cyclic voltammograms of LCCIa contain a quasi-reversible peak at 0.18V vs. SCE and an irreversible peak at 0.50V vs. SCE. We find that the replacement of the eleven amino acids a the C-terminus with a single cysteine residue $(i.e., \;LCCI{\rightarrow}LCCIa)$ influences the rate of heterogeneous electron transfer between an electrode and the copper containing active sites $(K_{het}\;for\;LCCI=1.0\times10^{-2}\;s^{-1}\;and\;K_{het}\;for\;LCCI_a= 1.0\;times10^{-1}\;s^{-1}\'at\;0.18V\;versus\;SCE\;and\;4.0\times10^{-2}\;s^{-1}\;at\;0.50V\; versus\;SCE)$. These results show for the first time that the change of the primary structure of a protein via site-directed mutagenesis influences both the redox potentials of the copper ions in the active site and the rate of heterogeneous electron transfer.