• Animal cells and systems
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• 제1권2호
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• pp.323-328
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• 1997

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.620-628
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• 2015

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFP_R9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP_R9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP_R9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFP_R9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP_R9 was indispensable for its antifungal action. The cDNA encoding for AfAFP_R9 was cloned from A. fumigatus R9 by RT-PCR and heterologously expressed in E. coli. The recombinant AfAFP_R9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFP_R9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.

• Applied Biological Chemistry
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• 제40권4호
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• pp.277-282
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• 1997

Acetolactate synthase (ALS) was partially purified from Escherichia coli MF2000/pTATX containing Arabidopsis thaliana ALS gene. The partially purified ALS was examined for its sensitivity toward various modifying reagents such as iodoacetic acid, iodoacetamide, N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (PCMB), and phenylglyoxal. It was found that PCMB inhibited the enzyme activity most strongly followed by DTNB and NEM. Since iodoacetic acid did not compete with substrate pyruvate, it appeared that cysteine is not involved in the substrate binding site. On the other hand, the substrate protected the enzyme partly from inactivation by phenylglyoxal, which might indicate interaction of arginine residue with the substrate. The partially purified enzyme was inhibited by end products, valine and isoleucine, but not by leucine. However, the ALS modified with PCMB led to potentiate the feedback inhibition of all end products. Additionally, derivatives of pyrimidyl sulfur benzoate, a candidate for a new herbicide for ALS, were examined for their inhibitory effects.

• The Korean Journal of Physiology
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• 제9권1호
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• pp.1-11
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• 1975

The present experiments were carried out to investigate the active center of sodium and potassium ion activated adenosine triphosphatase. An ATPase, activated by sodium ion Plus potassium ion in the presence of magnesium ion, and inhibited by ouabain, has been obtained from rabbit red cell ghosts. The ATPase activity was measured by inorganie phosphate released from ATP. From this values of the measured inorganic phosphate, the activity of ATPase was calculated. The following results were observed. 1. The activity of $(Na^++K^+)-ATPase$ is inhibited by ouabain. This effect may not be due to an effect on sulfhydryl groups, amino groups, carboxyl groups, imidazole groups and hydroxyl groups. 2. The $(Na^++K^+)$-activated enzyme system is inhibited by p-chloromercuribenzoate and by d nitroflurobenzene, and this effect may be due to an effect on sulfhydryl groups. These results indicate that the sulfhydryl groups is attached to sodium-potassium dependent adenosine triphosphate, an aspect of the pump. 3. The $(Na^++K^+)-activated$ enzyme system is inhibited by maleic anhydride and this inhibition is reversed by lysine. This Seems to indicate that the active center of this enzyme is the amino groups. 4. The $(Na^++K^+)$-activated enzyme system is inhibited by iodoacetamide and this inhibition is reversed by the simultaneous present of cysteine and aspartic acid in the suspension medium. This result indicates that this enzyme contains sulfhydryl groups and carboxyl groups. 5. The $(Na^++K^+)-ATPase$ activity is accelerated by adrenaline and this effect is abolished by aspartic acid. This effect of aspartic acid indicate that carboxyl group might be involved in the hydrolysis of ATP by the enzyme system. On the hydrolysis of ATP by the enzyme system. On the basis of these experiments it f·as suggested that the active center of $(Na^++K^+)-activated$ ATPase contains sulfhydryl groups, amino groups and carboxyl groups.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.106-111
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• 2005

For breeding of a new rootstock for eggplant production, somatic hybrids between two species, Solanum integrifolium and S. sanitwongsei were obtained through protoplast fusion. The former species has been commonly used for rootstock for eggplant production in Japan. Eggplants on these rootstocks are more productive than ungrafted plants, but are susceptible to bacterial wilt caused Ralstonia solanacearum. While the latter species is resistant, the growth of eggplants on this rootstock is rather slow and low yield. Protoplast of both species were isolated from cotyledons, and inactivated with iodoacetamide or UV-irradiation, then fused electrically. The fused products were then cultured. Regenerated plantlets were then transplanted on soil then maintained in a green house. The plants were classified into four groups. Those in the first group showed morphological characters intermediate of the parentalspecies. The plants bore fruit with viable seeds. The plants showed a chromosome number of 2n=48, the sum of those of the parental species, and are suggested to be symmetric fusion products. While plants in the other groupswas less vigorous and showed chromosome number 2n= 68 to 72 suggesting asymmetric fusion products by genomic in situ hybridization(GISH). Isozyme pattern of shikimate dehydrogenase (SKDH; EC 1.1.1.25), isocitrate dehydrogenase (IDH; EC 1.1.1.41) and phosphoglucomutase (PGM; EC 2.7.5.1) showed that 24 regenerated plants in three groups were somatic hybrids. Analysis of random amplified polymorphic DNA (RAPD) showed that 43 S. integrifolium-specific and 57 S. sanitwongsei-specific bands were all found in 24 plants. Both somatic hybrids and its S1 plants were found to be resistant to bacterial wilt, and eggplant grafted these plants using for rootstocks were more productive than grafted mother plants. Now, S1 progenies are used for commercial eggplant production in Osaka Prefecture.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.624-631
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• 2007

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

• BMB Reports
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• 제29권6호
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• pp.493-499
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• 1996

The biochemical properties of purified D-hydruxyisovalerate dehydrogenase from Fusarium sambucinum was elucidated. D-Hydroxyisovalerate dehydrogenase produced solely D-hydroxyisovalerate from 2-ketoisovalerate. The isoelectric point of the purified enzyme was 7.0. The enzyme was highly specific with 2-ketoisovalerate ($K_{m}=0.188$ mM, $V_{max}=8.814$ mmol/min mg) and 2-keto-3-methyl-n-valerate ($K_{m}=0.4$ mM, $V_{max}=1.851$ mmol/min mg) for the reductive reaction. This was also seen by comparing D-hydroxyisovalerate ($K_{m}=1.667$ mM, $V_{max}=0.407$ mmol/min mg) and D-hydroxy-3-methyl-n-valerate ($K_{m}=6.7$ mM, $V_{max}=0.648$ mmol/min mg) for the oxidative reaction. Thiol blocking reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromecuribenzoate inhibited about 80% of enzyme activity at 0.02 mM, 50 mM and 50 mM, respectively. The enzyme activity was also inhibited by the addition of 0.1 mM of various metal ions, such as $Fe^{2+}$ (67%), $Cu^{2+}$ (88%), $Zn^{2+}$ t (76%) and $Mg^{2+}$ (9%). The enzyme was stable over three months in 50 mM potassium phosphate buffer (pH 5~7) at $-80^{\circ}C$. However the purified enzyme lost 30% of its activity in the same buffer after 24 h at $4^{\circ}C$. The studies about thermal inactivation of D-hydroxyisovalerate dehydrogenase exhibit 209.2 kJ/M of activation enthalpy and 0.35 kJ/mol K of activation entropy.

• The Korean Journal of Zoology
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• 제31권3호
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• pp.157-164
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• 1988

SCK tumor cells were exposed to heat shock or several sulihydryl-reacting agents such as iodoacetamide(IAA), zinc chloride(Zn), and 2-mercaptoethanol(ME). Stress proteins induced by these agents were examined and the relationship between the induction of stress proteins and the production of abnormal proteins was discussed. Based on the present experiments, two classes of intracellular pathways for the induction of stress proteins were defined; one dependent on and the other independent of protein synthesis. The presence of cycloheximide during the induction period blocked the formation of stress proteins in the cells exposed to Zn or ME, but not in those exposed to heat shock or IAA.Therefore, stress protein seems to be induced either by denaturation of pre-existing mature proteins (e.g., heat shock or IAA) or by newly synthesized abnormal proteins(e.g., Zn or ME). In conclusion, it is ilkely that the production of abnormal proteins by stresses triggers stress protein induction. In addition, it was found that the cells exposed to IISP and GRP inducers simultaneously responded to more strong stress among several stresses encountered.

• Mass Spectrometry Letters
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• 제5권3호
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• pp.63-69
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• 2014

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

• Mass Spectrometry Letters
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• 제10권2호
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• pp.43-49
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• 2019

The aim of this study was conducted to develop an analytical method to determine the concentration of ceftiofur residue in eel, flatfish, and shrimp. For derivatization and extraction, the sample was hydrolyzed with dithioerythritol to produce desfuroylceftiofur, which was then derivatized by iodoacetamide to obtain desfuroylceftiofur acetamide. For purification, the process of solid phase extraction (Oasis HLB) was used. The target analytes were confirmed and quantified in $C_{18}$ column using liquid chromatography-tandem mass spectrometry with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as the mobile phase. The linearity of the standard calibration curve was confirmed by a correlation coefficient, $r^2>0.99$. The limit of quantification for ceftiofur was 0.002 mg/kg; the accuracy (expressed as the average recoveries) was 80.6-105%; the precision (expressed as the coefficient of variation) was below 6.3% at 0.015, 0.03, and 0.06 mg/kg. The validated method demonstrated high accuracy and acceptable sensitivity to meet the Codex guideline requirements. The developed method was tested using market samples. As a results, ceftiofur was detected in one sample. Therefore, it can be applied to the analysis of ceftiofur residues in fishery products.