Apoptosis유발물질의 세포독성에 대한 Iysosomal stabilizer의 보호효과

석대은,명평근 충남대학교 약학대학 의약품개발연구소 1996 藥學論文集 Vol.12 No.-

Protective action of lysosomal stabilizer against cytotoxicity of apoptosis-inducers such as dexamethasone or doxorubicin in L 1210 cells was examined. Pepstatin as an inhibitor of lysosomal protease, more efficient than leupeptin, raised the viability of cells exposed to dexamethazone or doxorubicin form 55 % to 67-76 %. It also showed some protection against cytotoxicity of shikonin, an inhibitor of topoisomerase-Ⅰ by enhancing the viability from 50.3 % to 78 %. Gentamycin, an inhibitor of lysosomal phospholipase, increased the viability (55%) of cells exposed to dexamethaslne or doxorubicin to 66 % and 60 %, respectively. Meanwhile, dibucaine as an aromatic lysosomal stabilizer failed to prevent the cytotoxicity of dexamethazone. Thus, since inhibitors of lysosomal hydrolases expressed a partial protection against the cytotoxicity of apoptosis-inducers, it is suggested that cytotoxicity of apoptosis-inducers might be partially ascribed to the activation of lysosomal hydrolases.

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+.

합성품 Peptide와의 비교실험을 통한 Bovine Thyroglobulin의 50K Polypeptide내에 존재하는 Diiodotyrosine-1375의 구조동정

류희문,석대은 충남대학교 약학대학 의약품개발연구소 2000 藥學論文集 Vol.16 No.-

A peptide fragment containing residues 1218-1591, prepared from thermolysin-mediated proteolysis of bovine thyroglobulin, was reduced by dithiothreitol and then treated with iodoacetic acid. The carboxymethylated peptide was digested with endoproteinase Asp-N, and fractionated by RP-HPLC. The fractions were analyzed by LC/ESI-MS for the monitor of a peptide with a hormonogenic site at Tyr-1375, and a fraction was found to contain to a peptide (residues 1366-1381) containing Tyr-1375. This observation was positively confirmed by the comparison with synthetic peptide, DVEEALAGK (diiodotyrosine) LAGRFA, which was produced from the oxidative iodination of DVEEALAGKYLAGRFA by lactoperoxidase employing KI and (H_2)(O_2)-generating system.

소 뇌막으로부터 5'-Nucleotidase의 효소적 용출에 대한 내재계면활성제의 효과

류희문,석대은 충남대학교 약학대학 의약품개발연구소 2000 藥學論文集 Vol.16 No.-

Glycosylphosphatidylinositol (GPI)-linked 5'-nucleotidase is released as both amphiphilic form (Amp-nucleotidase) and hydrophilic form (Hyd-nucleotidase) from bovine brain membrane. Exposure of brain membrane to deoxycholate, lysolecithin or monooleoylglycerol leads to a concentration-dependent release of Hyd-nucleotidase with deoxycholate being the most effective. Next, the brain GPI-PLD-catalyzed conversion of Amp-nucleotidase to Hyd-nucleotidase was investigated. The GPI-PLD-catalyzed conversion of required detergents, and among detergents used, monooleoylglycerol was the most effective for the enzymatic conversion. Monooleoylglycerol exhibited a concentration-dependent effect on GPI-PLD activity up to 0.3 mM, but its effect decreased at 1 mM. In addition, the combinational effect of monooleoylglycerol and lysolecithin on GPI-PLD action was not significant. Based on these results. it is suggested that the formation of Hyd-nucleotidase in brain tissue may be ascribed to the activities of GPI-PLC and GPI-PLD in the presence of bio-detergent.

Thiocholine ester 기질을 이용한 Acetylcholinesterase 활성부위의 구조특성

이천배,주은희,최수라,석대은,명평근 충남대학교 약학대학 의약품개발연구소 1999 藥學論文集 Vol.15 No.-

The inhibition pattern of three inhibitors(tacrine, decamethonium and propidium) on the hydrolysis of various thiocholine ester substrates by eel acetylcholinesterase was comparatively examined. When the substrate was acetylthiocholine, it showed a similar competitive inhibition by tacrine inhibitor, and a mixed type inhibition by decamethonium and propidium inhibitors. When the substrate was pentanoylthiocholine, it showed an uncompetitive inhibition by tacrine, and a noncompetitive inhibition by decamethonium. When the substrate was laurylthiocholine, it showed mixed type and uncompetitive inhibition by tacrine, and a competitive inhibition by decamethonium and propidium. Those results suggest that the active of acetylcholinesterase has the existence of hydorphobic site besides the anionic and esteratic site.

Thiocholine ester 기질을 이용한 Acetylcholinesterase 활성부위의 구조특성

이천배,주은희,최수라,석대은,명평근 충남대학교 기초과학연구소 1999 忠南科學硏究誌 Vol.26 No.1

The inhibition pattern of three inhibitors(tacrine, decamethonium and propidium) on the hydrolysis of various thiocholine ester substrates by eel acetylcholinesterase was comparatively examined. When the substrate was acetylthiocholine, it showed a similar competitive inhibition by tacrine inhibitor, and a mixed type inhibition by decamethonium and propidium inhibitors. When the substrate was pentanoylthiocholine, it showed an uncompetitive inhibition by tacrine, and a noncompetitive inhibition by decamethonium. When the substrate was laurylthiocholine, it showed mixed type and uncompetitive inhibition by tacrine, and a competitive inhibition by decamethonium and propidium. Those results suggest that the active site of acetylcholinesterase has the existence of hydrophobic site besides the anionic and esteratic site.

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.

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.

생약으로부터 산화적 결합 효소인 갑상선 peroxidase의 저해제 검색

이현정,장미영,김미리,배기환,석대은 충남대학교 약학대학 의약품개발연구소 1999 藥學論文集 Vol.15 No.-

Thyroid peroxidase is a biochemical target protein for the antithyroid drugs. Ethanol extracts from one hundred and thirty seven natural products were screened for the inhibition of thyroid peroxidase activity. Thyroid peroxidase was purified from porcine thyroids, and the inhibition of peroxidase activity was evaluated using guaiacol oxidation (C-C coupling) assay. Twenty one natural products expressed a remarkable inhibition (>50%) of peroxidase activity at 330㎍ solid weight/ml. The 50% inhibitory concentration (IC50) of 70% ethanol extract from six potent natural products ranged from 3.1 to 31.2㎍ solid weight/ml, in contrast to the range (0.33∼0.54㎍/ml) of IC50, values for catechin and epigallocatechin gallate as positive controls. Noteworthy, the extract of Camellia taliensis showed an irreversible inhibition of the enzyme. It is suggested that extract from some natural products such as Camellia taliensis, Rheum undulatum or Euphorbia pekinsis, exhibiting a potent inhibition of peroxidase activity, may be developed as sources of potent antithyroid agents.