• Title/Summary/Keyword: Iodoacetamide

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Purification and Characterization of an Intracellular Protease from Pseudomonas carboxydohydrogena (Pseudomonas charboxydohydrogena에서 분리 정제된 세포내 단백질 가수분해효소의 특성)

  • 이혜숙;김영민
    • Korean Journal of Microbiology
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    • v.29 no.3
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    • pp.167-171
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    • 1991
  • An intracellular protease from cells of Pseudomonas carboxydohydrogena grown on nutrient broth was purified to better than 95% homogeneity in five steps using azocaseine as a substrate. The molecular weight of the native enzyme was determined to be 125, 000. Sodium dodecyl sulfate-gel electrophoresis revealedat least two non-identical subunits of molecular weight 70, 000 and 56, 000. The enzyme activity was completely ingibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The enzyme was also inhibited by $Mg^{2+}$ , $Zn^{2+}$ , $Cd^{2+}$, $Cu^{2+}$ , and $Fe^{2+}$ , but was stimulated by iodoacetamide. Maximal reaction rate of the enzyme was observed at pH8.0 and 30.deg.C. The isoelectric point of the enzyme was found to be 7.5. The enzyme was unable to hydrolyze carbon monoxide dehydrogenase.

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Effect of IOA and Media on Production of Somatic Hybrid by the Asymmetric Protoplast Fusion Between Three Solanum Species

  • Kim Hag-Hyun;Kwon Soo-Jeong;Shin Un-Dong
    • Korean Journal of Plant Resources
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    • v.19 no.3
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    • pp.418-421
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    • 2006
  • In this study, we surveyed the influence of IOA (iodoacetamide) and media upon protoplast fusion for the efficient production of the somatic hybrid among S. sisymbriifolium and other Solanum species (S. intergrifolium and S. toxicarium). Regardless of a breed, as the IOA concentration increases, the cell division tends to decrease. As the influence of the media on the colony formation, we could get 5 colonies from the fusion of S. sisymbriifolium and IOA-treated S. integrifolium protoplast, but none was observed in other fusions. As a result of analyzing their IDH isozyme, we found a somatic hybrid in 2 objects.

Characterization of proteases of Toxoplasma gondii (Toxoplasma gondii에서 단백질 분해 효소의 특징)

  • Choe, Won-Yeong;Nam, Ho-U;Yun, Ji-Hye
    • Parasites, Hosts and Diseases
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    • v.27 no.3
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    • pp.161-170
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    • 1989
  • The proteases of Toxoplasma gcndii were purified partially and characterisrd for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteasfs working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion rxchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotorf. The protease working at pH 6.0 was inactivated by iodoacetamide with LDso of 10-5 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10-5 M and was Promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

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Degradation of human immunoglobulins and cytotoxicity on HeLa cells by live Trichomonas vaginalis (질편모충 단백질분해효소의 세포독성 및 인체면역글로불린 분해능)

  • 민득영;류재숙
    • Parasites, Hosts and Diseases
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    • v.35 no.1
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    • pp.39-46
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    • 1997
  • The present study was undertaken to determine whether live T. uaginnlis degrades human secretory IgA, serum If and IgG molecules. Human immunoglobulins were exposed to live trophozoites, parasite Iysate, and excretory-secretory product (ESP) of T ucginnlis. To determine the fragmentation of immunoglobulins, the reaction sample was subjected to SDS-PAGE and EITB, and peroxidase conjugated antihuman IgA and IgG were used as probes. Live trophozoites degraded secretory IgA, serum IgA and IgG, and degradation were pressed forward by the prolongation of the incubation time and by increasing the number of trichomonads respectively. Also the Iysates and ESP of trichomonads degraded IgA and IgG. The cysteine and serine proteinase inhibitors such as I-64, antipain, iodoacetic acid, iodoacetamide, TLCK reduced the ability of cleaving immunoglobulins. The proteinase activity and cytotoxicity of T. uaginnlis to HeLa cells were decreased when live T. vusinalis was treated with metallo-proteinase inhibitor as well as cysteine and serine proteinase inhibitors. These results suggest that proteinase secreted from live T ucginclis may play a part role in host pathogenesis by T. uosinnlis, and the cleaving ability of host immunoglobulins by the proteinase may contribute as a one of immune evasion mechanism for parasite survival in the host.

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Detection and Characterization of 45 kDa Platelet Activating Factor Acetylhydrolase in Cerebrospinal Fluid of Children with Meningitis

  • Moon, Tae-Chul;Kim, Mi-Suk;Lee, Su-Jeong;Lee, Tae-Yoon;Kwon, Soon-Hak;Baek, Suk-Hwan;Chang, Hyeun-Wook
    • Archives of Pharmacal Research
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    • v.26 no.7
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    • pp.554-558
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    • 2003
  • Platelet activating factor acetylhydrolase (PAF-AH) activity has been identified in cerebrospinal fluid (CSF) samples taken from children with meningitis. We reported that PAF-AH activity is significantly increased, by about 3 fold, in patients with meningitis compared to control subjects. Because of limited knowledge about this enzyme in CSF, we examined the biochemical properties of CSF PAF-AH. PAF-AH of CSF was calcium independent, showed a broad pH spectrum and was relatively heat stable. In addition, this enzyme activity was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), partially inhibited by p-bromophenacylbromide (p-BPB), uninhibited by iodoacetamide, and moderately stimulated by dithiothreitol (DTT). PAF-AH of CSF did not degrade phospholipid with a long chain fatty acyl group at sn-2 position. This enzyme hydrolyzed PAF and oxidatively modified phosphatidylcholine. Furthermore, we identified a monomeric polypeptide with a molecular weight of approximately 45 kDa by Western blot using human plasma PAF-AH antibody. These results suggested that plasma type PAF-AH activity exist in CSF taken from children with meningitis.

Chemical Modification of the Biodegradative Threonine Dehydratase from Serratia marcescens with Arginine and Lysine Modification Reagents

  • Choi, Byung-Bum;Kim, Soung-Soo
    • BMB Reports
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    • v.28 no.2
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    • pp.124-128
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    • 1995
  • Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.

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The Oxidative Modification of COL6A1 in Membrane Proteins of Ovarian Cancer Patients

  • Yang, Hee-Young;Lee, Tae-Hoon
    • Reproductive and Developmental Biology
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    • v.36 no.1
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    • pp.39-47
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    • 2012
  • Ovarian cancer is the most lethal gynecological malignancy, and specific biomarkers are important needed to improve diagnosis, prognosis, and to forecast and monitor treatment efficiency. There are a lot of pathological factors, including reactive oxygen species (ROS), involved in the process of cancer initiation and progression. The oxidative modification of proteins by ROS is implicated in the etiology or progression of disorders and diseases. In this study, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) revealed that a variety of proteins were differentially oxidized between normal and tumor tissues of ovarian cancer patients. To identify cysteine oxidation-sensitive proteins in ovarian cancer patients, we performed comparative analysis by nano-UPLC-$MS^E$ shotgun proteomics. We found oxidation-sensitive 22 proteins from 41 peptides containing cysteine oxidation. Using Ingenuity program, these proteins identified were established with canonical network related to cytoskeletal network, cellular organization and maintenance, and metabolism. Among oxidation-sensitive proteins, the modification pattern of Collagen alpha-1(VI) chain (COL6A1) was firstly confirmed between normal and tumor tissues of patients by 2-DE western blotting. This result suggested that COL6A1 might have cysteine oxidative modification in tumor tissue of ovarian cancer patients.

Purification and Characterization of Adenosine deaminase from Aspergillus oryzae (Aspergillus oryzae에서 Adenosine Deaminase의 정제와 특성)

  • Choi, Hye-Seon
    • Korean Journal of Microbiology
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    • v.31 no.1
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    • pp.54-62
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    • 1993
  • Intracellular adenosine deaminase (ADA) from Aspergillus oryzae was purified using ammonium sulfate fractionation, a DEAE-Sephadex A-50 anion exchange chromatography, an ultrafiltration using a PM 10 membrane and two times of Sephadex G-100 gel filtration chromatography. The enzyme was purified 151 fold with a 9% recovery. Purified enzyme gave a single protein band with a molecular weight of 105,000 delton. The enzyme was reasonably stable. The enzyme activity was kept even after 1 hr incubation at 55.deg.C, but decreased significantly at 60.deg.C. The pH optimum was found to be from 6.5 to 7.5. Among tested compounds, the substrate activity was found with adenosine, adenine arainofuranoside, formymcin A, 2'-deoxyadenosine, 3'-deoxyadenosine, 2', 3'-isopropylidene adenosine, 2,6-diaminopurine deoxyriboside, .betha.-nicotinamide adenine dinucleotide (reduced form), 6-chloropurine riboside, 2'-adenine monophosphate (AMP), 3'-AMP and 5'-AMP. The values of Km of adenosine and 2'-deoxyadenosine were calculated to be 500 and .$710\mu$m, respectively. ADA was sensitivite to $Zn^{2+}$, $^Cu{2+}$ and $Fe^{3+}$, p-chloromercuribenzoate and mersalyl acid inactivated the enzyme. The activity of enzyme was not changed when ADA was incubated with dithiothreititol, 2-mercaptoethanol, N-ethylmaleimide, iodoacetic acid and iodoacetamide.

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Purification and Characterization of an α-D-Galactosidase from Grape Berry

  • Kang, Han-Chul;Kim, Tae-Su
    • Journal of Applied Biological Chemistry
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    • v.43 no.3
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    • pp.141-146
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    • 2000
  • Glycosidase activities were tested from the grape berries, Vitis labruscana B. Takasumi. Among various glycosidases, $\alpha$-D-galactosidase was found to be the most active in the flesh and other glycosidases were considerably active in the order of the following: $\alpha$-D-mannosidase>$\alpha$-D-glucosidase>$\beta$-D-glucosidase>$\beta$-D-galactosidase. In the seeds, $\alpha$-D-glucosidase activity was the highest and other glycosidases such as $\alpha$-D-galactosidase, $\beta$-D-glucosidase, and $\beta$-D-galactosidase were still significantly active. The $\alpha$-D-galactosidase in the grape flesh was purified over 83-folds through salting-out with $(NH_4)_2SO_4$ and a series of chromatographies employing Sephadex G-50, Octyl-Sepharose, Q-Sepha- rose, and Biogel P-100. The enzyme was a monomer of 45 kDs as determined through SDS-PAGE and Sephacryl S-200 chromatography. The purified enzyme showed a preference of $\alpha$-D-galactose to $\beta$-D-galactose as a substrate about 5.4 times. Sulfhydryl specific reagents such as N-ethylmaleimide and iodoacetamide significantly inhibited the enzyme activity to the extents of 48 and 52% of its initial activity, respectively. The optimumpH range of $\alpha$-D-galactosidase was around 6.5-7.0. The enzyme activity increased by 46% in the presence of 1mM $Fe^{2+}$.

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Antioxidants Effects and Roles of Endogenous Sulfhydryls in the Gastric Mucosal Protection of Puerariae Radix (갈근(葛根)의 항산화작용에 의한 위점막 보호효과와 내인성 Sulfhydryl 화합물의 영향)

  • Choi, Ho-Jeong;Shin, Heung-Mook
    • Journal of Oriental Physiology
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    • v.14 no.2 s.20
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    • pp.1-9
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    • 1999
  • Ethanol induces compoundhemorrhagic gastric lesions and causes a dose-dependent decrease in the concentration of endogenous nonprotein sulfhydryls in rat gastric mucosa. Sulfhydryl-containing drugs protect rats from ethanol - induced gastric lesions. Based on this findings, we investigated the involvement of sulfhydryl compounds in the antioxidant effects of Puerariae radix, a traditional herbal medicine, against ethanol - induced gastric lesions in the absence and presence of iodoacetamide(IDA. sulfhydryl blocking agent) in rats. respectively. Because of the known role of sulfhydryls in gastric cytoprotection, its role in gastric antioxidation was of intrest. In vitro, Puerariae radix extract(PRE) reduced linoleic acid autooxidation and exert DPPH radical scavenging effect. In vivo. PRE increased antioxidants(SOD, catalase. GSH) and reduced lipid peroxide level in ethanol-induced gastric mucosal lesions. But treatment with PRE plus IDA significantly inhibit the antioxidant effects such as SOD and GSH but did not affect catalase levels. These results suggest that Puerariae radix may play roles in the gastric cytoprotection through antioxidant effects and increase of SOD activity and GSH level are dependent of endogenous sulfhydryls.

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