Simple Isolation of DNA from the Tissue Homogenate by Acridinium Affinity Chromatography

석대은,정창희,김윤배,정윤수,Sok, Dai-Eun,Jung, Chang-Hee,Kim, Yun-Bae,Chung, Yun-Su Korean Society for Biochemistry and Molecular Biol 1990 한국생화학회지 Vol.23 No.3

고염농도(1.4 M NaCl)의 어류 근조직 균질액을 아크리디늄 친화성 컬럼에 흡착시킨 후 2M $MgCl_2$로 용출시켜 고순도의 DNA ($A_{260}/A_{280}$?2.0)를 얻었다. 이 컬럼에 흡착된 DNA는 0.5M $MgCl_2$로도 용출되었으나 2M NaCl로는 용출되지 않았다. 한편, DNase-I 처리로 친화성 컬럼상의 DNA의 크로마토그래피 유형이 달라졌으며, 이 친화성 컬럼은 단일 및 이중 나선형 DNA 모두에 좋은 결합 친화성을 가진 것으로 여겨진다. 따라서, 이 방법은 고이온강도에서 생조직 균질액으로부터 DNA를 분리하는데 적합할 것이다. The homogenate of fish muscle tissue in high salt concentration (1.4 M NaCl) was applied on the acridinium affinity column, and the elution of the column with 2 M $MgCl_2$ produced the material, which turned out to be a highly pure DNA ($A_{260}/A_{280}$?2.0). The DNA bound on the column was desorbed easily with 0.5 M $MgCl_2$, but not even with 2 M NaCl. Separately, the treatment with DNase-I altered the chromatographic profile of DNA on the affinity column, and the affinity column seems to express good binding affinity toward both single and double stranded DNAs. This method would be suitable for the separation of DNA from crude tissue homogenates in high ionic strength.

5 , 15 - diHETE 의 주요 생합성 경로 : 아리키돈산으로부터 15 - HPETE를 거쳐 5 , 15 - diHETE로의 전환과정

석대은,찰스죤시 ( Dai Eun Sok,Charles J . Sih ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

In both RBL-1 cells and human leukocytes, the calcium ionophore A 23187 enhanced the conversion of 15(S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) to 5,15-diHETE by Ca^(++)-dependent 5-lipoxygenase (5-LPO). Interestingly, the amount of 5,15-diHETE formed from the incubation of 15(S)-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with RBL-1 cells was similar to that generated from the incubation of 15-HETE with RBL-1 cells in the presence of A23187, suggesting that the 5-LPO-catalyzed conversion of 15-HPETE into 5,15-diHETE is independent of calcium ion. As a precursorial substrate for transformation to 5,15-diHETE in human leukocytes, 15-HPETE was found to be three times more efficient than 5-HPETE (5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid). When 15-HETE, a strong inhibitor of lipoxygenation of arachidonic acid at C-5 or C-12, was added to the leukocytes suspension containing arachidonic acid and A 23187, the formation of 5,15-diHETE from arachidonic acid was increased to a great extent ($gt;3 times). Thus, the formation of 5,15-diHETE from arachidonic acid via 15-HPETE, which is Ca^(++)-independent and insensitive to 15-HETE, is supposed to be the more favorable pathway for the biosynthesis of 5,15-diHETE in the intact cells.

대두 lipoxygenase 에 의한 아라키돈산으로부터 lipoxins 으로서 전환

석대은,피택산,정창희,정윤수,강정부 ( Dai Eun Sok,Taek San Phi,Chang Hee Jung,Yun Su Chung,Jung Bu Kang ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2

The exposure of arachidonic acid to soybean lipoxygenase led to the formation of polar products showing intense absorption at 300-301 nm. These products were found to be lipoxin A and B isomers, based on UV spectrum, HPLC and GC/MS analyses. This method, convenient to prepare hundreds of micrograms of lipoxin A and B isomers, would be useful for the study on the biosynthesis and the biological activity of lipoxins.

A Favorable Major Biosynthetic Pathway of 5(S), 15(S)-dihydroxy-6, 13-trans-8, 11-cis-icosatetraenoic acid (5,15-diHETE) from Arachidonic acid.

석대은,찰스 죤시,Sok, Dai-Eun,Sih, Charles J. 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

In both RBL-1 cells and human leukocytes, the calcium ionophore A 23187 enhanced the conversion of 15(S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) to 5,15-diHETE by $Ca^{++}$-dependent 5-lipoxygenase (5-LPO). Interestingly, the amount of 5,15-diHETE formed from the incubation of 15(S)-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with RBL-1 cells was similar to that generated from the incubation of 15-HETE with RBL-1 cells in the presence of A23187, suggesting that the 5-LPO-catalyzed conversion of 15-HPETE into 5,15-diHETE is independent of calcium ion. As a precursorial substrate for transformation to 5,15-diHETE in human leukocytes, 15-HPETE was found to be three times more efficient than 5-HPETE (5-hydroperoxy-6- trans-8,1l,14-cis-icosatetraenoic acid). When 15-HETE, a strong inhibitor of lipoxygenation of arachidonic acid at C-5 or C-12, was added to the leukocytes suspension containing arachidonic acid and A 23187, the formation of 5,15-diHETE from arachidonic acid was increased to a great extent (>3 times). Thus, the formation of 5,15-diHETE from arachidonic acid via 15-HPETE, which is $Ca^{++}$-independent and insensitive to 15-HETE, is supposed to be the more favorable pathway for the biosynthesis of 5,15-diHETE in the intact cells. 쥐종양세포 (RBL-1 cells)와 인체 백혈구내에서 calcium ionophore A 23187은 칼슘이온 의존성 리폭시게나제(5-LPO)를 활성화시켜 15-HETE로부터 5, 15-diHETE의 생성을 크게 증가시켰다. 흥미롭게도 15-HPETE로부터 5, 15-diHETE로의 전환은 calcium ionophore의 영향과 무관하였으므로, 15-HPETE는 5, 15-diHETE의 생합성을 위한 효과적인 전구체임을 알 수 있었다. 별도의 비교실험에서 15-HPETE는 5-HPETE보다 3배 이상 더 효과적인 전구체로 밝혀졌다. 한편, 아라키돈산의 5번, 12번 위치의 지방산 산화(lipoxygenation)를 방지하기 위한 선택적 저해제로 15-HETE를 백혈구 세포반응액에 첨가했을 때, 아라키돈산에서 생성되는 5, 15-diHET의 양은 3배 이상 증가하였는데, 이 결과는 아라키돈산의 15번 탄소위치에서 지방산 산화반응이 선택적으로 이루어짐을 의미하는 것이다. 따라서, 아라키돈산에서 15-HPETE를 거쳐 5, 1 5-diHETE로의 전환과정은 칼슘이온 비의존성이고, 15-HETE 저해에 무관한 효소과정으로서, 5, 15-diHPETE 생성의 주요생합성 과정으로 사료된다.

Soybean Lipoxygenase-Catalyzed Conversion of Arachidonic Acid into Lipoxins

석대은,피택산,정창희,정윤수,강정부,Sok, Dai-Eun,Phi, Taek-San,Jung, Chang-Hee,Chung, Yun-Su,Kang, Jung-Bu 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2

아라키돈산에 대두 lipoxygenase를 작용시켰을때, 300-301 nm에서 강한 흡수를 나타내는 극성 물질들이 형성되었다. 이 물질들을 UV스펙트럼, HPLC, GC/MS에 의한 분석 결과, lipoxin A와 lipoxin B 이성체로 확인되었다. 이 방법은 수백 마이크로그람의 liposin A 및 B 이성체를 얻는데 편리한 것으로, lipoxins의 생합성과 생리적 활성을 연구하는데 유용할 것으로 사료된다. The exposure of arachidonic acid to soybean lipoxygenase led to the formation of polar products showing intense absorption at 300-301 nm. These products were found to be lipoxin A and B isomers, based on UV spectrum, HPLC and GC/MS analyses. This method, convenient to prepare hundreds of micrograms of lipoxin A and B isomers, would be useful for the study on the biosynthesis and the biological activity of lipoxins.

브로콜리 용매추출물의 Bioactive Organosulfur 화합물의 분리 및 동정

석대은(Dai-Eun Sok),김진희(Jin Hee Kim),김미리(Mee Ree Kim) 한국식품영양과학회 2003 한국식품영양과학회지 Vol.32 No.3

브로콜리 중의 bioactive organosulfur phytochemicals를 분석하기 위해 브로콜리를 용매추출하여 GC/MSD에 의해 분리동정하였다. 브로콜리에 함유된 phytochemicals 중에서 isothiocyanate류가 40.42%로 대부분을 차지하였으며, glucosinolate 분해산물인 nitrile류는 5.12%로 isothiocyanate류에 비해 적었고, sulfide류는 0.84%로 매우 적었다. 분리 동정된 isothiocyanate류의 종류로는 3-butenyl, 4-methylthiobutyl, 4-methylthio-3-butenyl, 5-methylthiopentyl, 2-phenylethyl, 3-methylsulfinyl propyl 및 4-methylsulfinylbutyl isothiocyanate의 7종이었다. Isothiocyanate류 중에서 3-butenyl isothiocynate는 22.05%로 가장 많았으며, 그 다음으로 sulforaphane 16.50%로 2종의 isothiocyanate가 대부분을 차지하였다. Nitrile의 종류는 4-methylthiobutyl, 5-methylthiopentyl, 2-phenylethyl 및 4-methylsulfinylbutyl nitrile의 6종류가 확인되었다. 한편, 동정된 sulfide 종류로는 dimethyl disulfide, dimethyl trisulfide 및 dimethyl tetrasulfide이었다. Bioactive organosulfur phytochemicals were isolated from fresh broccoli using methlylene chloride as an extract solvent, and identified by GC/MSD analyses. Major organosulfur phytochemicals of broccoli extract were found to be isothiocyanates, which constitute 40.42% of total phytochemicals. The isothiocyanates from broccoli extract were identified to be 3-butenyl, 4-methyl thiobutyl, 4-methylthio-3-butenyl, 5-methylthiopentyl, 2-phenylethyl, 3-methyl sulfinyl propyl, and 4-methylsulfinylbutyl isothiocyanates, of which major isothio-cyanates were 3-butenyl isothiocyanate and 4-methylsulfinylbutyl isothiocyanate, constituting about 38.55% of total isothiocyanates present in the solvent extract. Also, nitriles, corresponding to products from enzymatic hydrolysis of glucosinolates were identified as 4-methylthiobutyl, 5-methyl thiopentyl, 2-phenylethyl and 4-methylsulfinylbutyl nitrile. In addition, three sulfides were identified as dimethyl disulfide, dimethyl trisulfide and dimethyl tetrasulfide.

외부화학물질(살충제)의 새로운 생체대사(유황산화)를 위한 모델

석대은 ( Dai Eun Sok ),김용식 ( Yong Sik Kim ),김광진 ( Kwang Jin Kim ),양영태 ( Young Tae Yang ),최정수 ( Jeong Soo Choi ) 국군의무사령부 1983 대한군진의학학술지 Vol.14 No.1

비가역적 살충제의 확인을 위한 고정화된 콜린에스테라제의 응용

석대은 ( Dai Eun Sok ),이중기 ( Choong Ki Lee ),이현재 ( Hyun Jae Lee ) 국군의무사령부 1985 대한군진의학학술지 Vol.16 No.1

생쥐 뇌의 Acetylcholinesterase 의 정제 및 특성

조영,석대은,고동성 ( Young Cho,Dai Eun Sok,Thong Sung Ko ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.5

Acetylcholinesterase (AChE) was solubilized from mouse brain membrane by 10% Triton X 100 treatment, and partially purified by DEAF-Sephacel chromatography plus affinity chromatography employing N-methylacridinium as the affinity ligand. The molecular weight of the partially purified enzyme was determined by high performance liquid chromatography (HPLC). It was revealed that the purified enzyme exists as two associated forms of molecular weight of 339,000 and 609,000. Concanavalin A affinity chromatography shows that this enzyme is a type of a hydrophobic glycoprotein that is bound to the membrane. K_m value and specific activity of the purified enzyme which possessed the optimum pH of 9.0 were found to be 46 μM for acetylcholine and 215.6 units/㎎, respectively. Inhibition studies show that the enzyme is inhibited strongly by hydrophobic compounds containing a cationic group.

대두 리폭시게나제 효소와 아라키돈산의 반응중 자기불활성화의 요인에 관련된 중간체 생성물

김미리,석대은 ( Mee Ree Kim,Dai Eun Sok ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.4

Gradual decrease of soybean lipoxygenase activity was observed during the prior incubation with arachidonic acid, while preincubation with linoleic acid or 11,14,17-eicosatrienoic acid had no effect on lipoxygenase activity. Among the lipoxygenation products of arachidonic acid, only 15(S)-hydroperoxy eicosatetraenoic acid (15(S)-HPETE) inhibited lipoxygenase in a time-dependent manner. Either further oxygenation products of 15(S)-HPETE or non-enzymatic decomposition products of 15(S)-HPETE had no significant inhibitory effect. The observations that the inhibitory effect of 15(S)-HPETE was transient, and not affected by hydroxy radical scavangers support the assumption that the unstable lipid intermediates produced from further conversion of 15(S)-HPETE may be responsible for the irreversible inhibition of lipoxygenase. In support of the above assumption, 13(S)-hydroperoxy-6, 9, 11-octadecatrienoic acid, which can be further lipoxygenated was also found to inactivate lipoxygenase effectively. However, 13(S)-hydroperoxy-9,11-octadecadienoic acid or 13(S)-hydroperoxy-9,11,15-octadecatrienoic acid was not effective.