소 뇌 조직으로부터 5'-Nucleotidase의 정제 및 특성규명

류희문,석대은 충남대학교 약학대학 의약품개발연구소 2002 藥學論文集 Vol.17 No.-

5'-Nucleotidase, bound to brain membranes as a glycosylphosphatidyl-inositol (GPI)-anchored protein, is responsible for the conversion of adenosine-5'-monophosphate into adenosine, which is an agonist in adenosine receptor signalling. Here, 5'-nucleotidase was isolated from bovine brain using PI-PLC treatment, and purified by concanavalin A sepharose chromatography, DEAE-sephacel chromatography, and finally AMP affinity chromatography. For higher yield of enzyme purification, Zn^2+ was Included in the elution buffer in DEAE-sephacel chromatography. Especially, NaCl was more favorable than MgCl_2 for the elution of 5'-nucleotidase, proper for inactivation study, from AMP affinity column. The purified 5'-nucleotidase was relatively pure on SDS-PAGE analysis, showing a specific activity of 30.27 μmole/min/㎎ (purification fold 19,000 fold). The purified enzyme, possessing a K_m value of 44μM and an optimum pH of 7.5, was inhibited competitively by ATP (K_i, 12 μM), and uncompetitively by cysteine (K_i, 0.32 mM). In addition, the enzyme was activated slighty (1.5-folds) by Mg^2+.

Cumene hydroperoxide에 의한 paraoxonase 1의 산화적 불활성화에 대한 보호 방안

스, 위엥쥐,류희문,김주령,석대은 충남대학교 약학대학 의약품개발연구소 2003 藥學論文集 Vol.18 No.-

Paraoxonase 1 (PON1), an enzyme associated with high density lipoprotein (HDL), is known to protect low density lipoprotein (LDL) from lipid peroxidation involving copper ion. However, Paraoxonase 1 activity was observed to decrease during LDL oxidation. Here, the inactivation of PON1 by various peroxides was examined. Paraoxonase 1, purified from human plasma, was subjected to cumene hydroperoxide, hydrogen peroxide or tert-butyl hydroperoxide. PON activity, based on the hydrolysis of phenyl acetate, decreased by approximately 40 and 38 %, respectively, after the exposure to 2mM cumene hydroperoxide and hydrogen peroxide, while tert-butyl hydroperoxide had no remarkable inhibitory effect. Next, the compounds capable of preventing against cumene hydroperoxide-induced inactivation of PONl were screened. While quercetin or phenyl acetate failed to protect PON1, lauric acid or calcium chloride was found to protect PONl from cumene hydroperoxide-induced inactivation. Especially, lauric acid appeared to show the greater protection than the other fatty acids tested. In further study, lauric acid showed a dose-dependent protection with an E& value of around 35 μM. Based on these results, It is proposed that the alky hydroperoxide-induced inactivation of Paraoxonase 1 can be prevented by a proper fatty acid recipe.

효소 Lipoxygenase의 신규기질 : Acylglycerol, acylethanolamide, lysophospholipids 및 phospholipids

황룡쌍,류희문,박천호,석대은 충남대학교 약학대학 의약품개발연구소 2007 藥學論文集 Vol.22 No.-

Lipoxygenase belongs to a diverse family of nonheme ferroproteins that oxygenate polyenoic fatty acids containing 1,4-pentadiene structure to form their corresponding hydroperoxy derivatives. Lipoxygenases (LOXs), widely distributed in animals and plants, have a key function in the formation of biologically active substances from pulyunsaturated fatty acids. Generally, free polyunsaturated fatty acids, liberated from membrane phospholipids via phospholipase-catalyzed hydrolysis, are used as substrates for LOXs. Although it is acknowledged that free polyunsaturated fatty acids are preferred to phospholipids or triglycerides as substrates, there have been recent reports that mammalian enzymes can oxidize certain phospholipids. Especially, reticulocyte LOX (15-LOX) leukocyte 15-LOX, leukocyte LOX (12-LOX) can oxygenate complex substrates such as phospholipids and biomembranes. In addition, acylglycerol and acylethanolamide are utilized by lipoxygeanse as well as cycoloxygenase; the latter enzyme contributes to generation of bioactive prostanoids derivative. Furthermore, lysophosphatidylcholine or lysophosphatidic acid containing linoleoyl or arachidonoyl moieties are known to be oxygenated by reticulocyte LOX, leukocyte 15-LOX or leukocyte-type 12-LOX; oxygenated lysophospholipids can play a carrier role in transporting oxygenated derivatives. Thus, the use of various lipid substrates as new substrates for lipoxygenase may extend the physiological roles of those lipids containing unsaturated fatty acyl chains.

인체 혈장 Paraoxonase의 산화적 불활성화

위엥쥐스,김주령,정태숙,류희문,석대은 충남대학교 약학대학 의약품개발연구소 2002 藥學論文集 Vol.17 No.-

Paraoxonase (PON), an enzyme associated with high density lipoprotein (HDL), is known to protect low density lipoprotein (LDL) from lipid peroxidation involving copper ion. However, PON activity was observed to decrease during LDL oxidation. Here, we attempted to elucidate the possible mechanism for the inactivation of PON. PON was partially purified from human plasma, and subjected to various oxidant systems. PON activity, based on the hydrolysis of phenyl acetate, decreased slightly after the exposure to H_2O_2 or ascorbate, while oxidants such as peroxynitrite or HOCl had no remarkable effect. Inclusion of Cu^2+ in the incubation with ascorbate (0.3∼1 mM) led to a rapid decrease of activity in a time-and concentration-dependent manner. In comparison, ascorbate/Cu^2+ system was much more effective than ascorbate/Fe^2+ system in inactivating PON. A further study indicates that general hydroxyl radical scavengers such as mannitol, ethanol or benzoate failed to prevent the PON inactivation. Based on these results, it is proposed that the PON inactivation during LDL oxidation may be ascribed mainly to the Cu^2+-catalyzed oxidation.

Synthesis and Characterization of Soluble Polyimides Containing PolyaIicycIic Structures

Wen Xi Huang,이미혜,최길영 한국고분자학회 1997 한국고분자학회 학술대회 연구논문 초록집 Vol.1997 No.4

Multimerization of Bovine Thyroglobulin, Partially Unfolded or Partially Unfolded/Reduced; Involvement of Protein Disulfide Isomerase and Glutathionylated Disulfide Linkage

Xi-Wen Liu,Dai-Eun Sok 대한약학회 2004 Archives of Pharmacal Research Vol.27 No.12

Fate of the nascent thyrolglobulin (Tg) molecule is characterized by multimerization. To establish the formation of Tg multimers, the partially unfolded/reduced Tg or deoxycholate-treated/ reduced Tg was subjected to protein disulfide isomerase (PDI)-mediated multimerization. Oxidized glutathione/PDI-mediated formation of multimeric Tg forms, requiring at least an equivalent molar ratio of PDI/Tg monomer, decreased with increasing concentration of reduced glutathione (GSH), suggesting the oxidizing role of PDI. Additional support was obtained when PDI alone, at a PDI/Tg molar ratio of 0.3, expressed a rapid multimerization. Independently, the exposure of partially unfolded Tg to GSH resulted in Tg multimerization, enhanced by PDI, according to thiol-disulfide exchange. Though to a lower extent, a similar result was observed with the dimerization of deoxycholate-pretreated Tg monomer. Consequently, it is implied that intermolecular disulfide linkage may be facilitated at a limited region of unfolded Tg. In an attempt to examine the multimerization site, the cysteine residue-rich fragments of the Tg were subjected to GSH-induced multimerization; a 50 kDa fragment, containing three vicinal dithiols, was multimerized, while an N-terminal domain was not. Present results suggest that the oxidase as well as isomerase function of PDI may be involved in the multimerization of partially unfolded Tg or deoxycholate-treated Tg.

Reductive Depolymerization of Bovine Thyroglobulin Multimers via Enzymatic Reduction of Protein Disulfide and Glutathionylated Mixed Disulfide Linkages

Xi-Wen Liu,Dai-Eun Sok 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.9

The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxycholate- treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.

Multimerization of Bovine Thyroglobulin, Partially Unfolded or Partially Unfolded/Reduced; Involvement of Protein Disulfide Isomerase and Glutathionylated Disulfide Linkage

Liu, Xi-Wen,Sok , Dai-Eun The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.12

Fate of the nascent thyrolglobulin (Tg) molecule is characterized by multimerization. To establish the formation of Tg multimers, the partially unfolded/reduced Tg or deoxycholate-treated/ reduced Tg was subjected to protein disulfide isomerase (PDI)-mediated multimerization. Oxidized glutathione/PDI-mediated formation of multimeric Tg forms, requiring at least an equivalent molar ratio of PDI/Tg monomer, decreased with increasing concentration of reduced glutathione (GSH), suggesting the oxidizing role of PDI. Additional support was obtained when PDI alone, at a PDI/Tg molar ratio of 0.3, expressed a rapid multimerization. Independently, the exposure of partially unfolded Tg to GSH resulted in Tg multimerization, enhanced by PDI, according to thiol-disulfide exchange. Though to a lower extent, a similar result was observed with the dimerization of deoxycholate-pretreated Tg monomer. Consequently, it is implied that intermolecular disulfide linkage may be facilitated at a limited region of unfolded Tg. In an attempt to examine the multimerization site, the cysteine residue-rich fragments of the Tg were subjected to GSH-induced multimerization; a 50 kDa fragment, containing three vicinal dithiols, was multimerized, while an N-terminal domain was not. Present results suggest that the oxidase as well as isomerase function of PDI may be involved in the multimerization of partially unfolded Tg or deoxycholate-treated Tg.

Reductive Depolymerization of Bovine Thyroglobulin Multimers via Enzymatic Reduction of Protein Disulfide and Glutathiony­lated Mixed Disulfide Linkages

Liu Xi-Wen,Sok Dai-Eun The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.9

The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxy­cholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.

Role of Protein Disulfide Isomerase in Molecular Fate of Thyroglobulin and its Regulation by Endogenous Oxidants and Reductants

Liu, Xi-Wen,Sok, Dai-Eun The Pharmaceutical Society of Korea 2002 Archives of Pharmacal Research Vol.25 No.4

The molecular fate of thyroglobulin (Tg) is controlled by oligomerization, a means of storing Tg at high concentrations, and deoligomerization. The oligomerization of bovine Tg are intermolecular reactions that occur through oxidative processes, such as disulfide and dityrosine formation, as well as isopeptide formation; disulfide formation is primarily responsible for Tg oligomerization. Here, the protein disulfide isomerase (PDI) and/or peroxidase-induced oligomerization of unfolded thyroglobulins, which were prepared by treating bovine Tg with heat, urea or thiol/urea, was investigated using SDS-PAGE analyses. In addition, the enzymatic oligomerization was compared with non-enzymatic oligomerization. The thermally-induced oilgomerization of Tg, dependent on glutathione redox state, was affected by the ionic strength or the presence of a surfactant. Meanwhile, PDI-catalyzed oligomerization, time and pH-dependent, was the most remarkable with unfolded/reduced Tg, which was prepared from a treatment with urea/DTT, while the thermally-unfolded Tg was less sensitive. Similarly, the oligomerization of unfolded/reduced Tg was also mediated by peroxidase. However, PDI showed no remarkable effect on the peroxidase-mediated oligomerization of either the unfolded or unfolded/reduced Tg. Additionally, the reductive deoligomerization of oligomeric Tg was exerted by PDI in an excessively reducing state. Based on these results, it is proposed that PDI catalyzes the oligomerization of Tg through the disulfide linkage and its deoligomerization in the molecular fate, and this process may require a specific molecular form of Tg, optimally unfolded/reduced, in a proper redox state.