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Human tyrosinase 유전자의 대장균에서의 발현
박소영,공광훈,박희중 中央大學校 遺傳工學硏究所 1997 遺傳工學硏究論集 Vol.10 No.1
Tyrosinase는 활성부위에 한 쌍의 구리이온을 함유하고 있는 metalloprotein으로 멜라닌 생합성에 중요한 역할을 한다. Human tyrosinase의 메카니즘을 연구하기 위해 human tyrosinase 유전자에 대한 대장균에서의 대량발현계를 구축하였다. Plasmid pRHOHT2 (Shibahara et al, 1988. Tohoku J. Exp. Med. 156. 403)로부터 human tyrosinase cDNA sequence는 polymerase chain reaction에 의해 증폭되었으며, 발현벡터 pGEX-KG와 pTrcHis에 subcloning 되었다. 이렇게 작성된 plasmid pKG-Tyrosinase와 pHis-Tyrosinase를 대장균주 MV1190에 형질전환시켜, 유도제 isopropyl- β-D-thiogalactopyranoside로 human tyrosinase를 대량발현시켰다. 발현된 tyrosinase는 GSH affinity column과 immobilized metal affinity column으로 정제하였고, 정제된 재조합 tyrosinase는 SDS-gel 전기영동상에서 각각 84,000과 66,000 정도를 나타내었다. 또한 정제된 재조합 tyrosinase는 dopa oxidation 활성을 나타내었다. Tyrosinase is a bifunctional copper-containing metalloprotein and play a central role and of melanin biosynthesis. In the study, we constructed several E. coil-expression system of human tyrosinase using a cDNA encoding human tyrosinase for its mechanism study. Human tyrosinase cDNA sequence, plasmid pRHOHT2 (Shibahara et al. 1988, Tohoku J. Exp. Med. 156, 403), was amplified by the polymerase chain reaction and was subcloned with pGEX-KG and pTrcHis expression vector. These recombinant tyrosinases were produced by induction with isopropyl-β-D-thiogalactopyranoside. Overexpressed human tyrosinases were purified by GSH affinity column and immobilized metal affinity column. The molecular weights of the purified recombinant tyrosinases determined by SDS-polyacrylamide gel electrophoresis was about 84,000 (pKG-Tyrosinase) and 66,000 (pHis-Tyrosinase), respectively. These enzymes also showed dopa oxidation activity.
Human Glutathione S-transferase의 Tyr108 잔기의 역할에 관한 연구
박희중,공광훈 中央大學校 遺傳工學硏究所 1999 遺傳工學硏究論集 Vol.12 No.1
Glutathione S-transferase(GST, EC 2.5. 1.18)의 기질 결합부위에 대한 연구를 위하여 human GST P 1-1의 친전자성 기질 결합부위 (H-site)로 알려지고 있는 Tyr108 잔기에 대하여 부위특이적 변이법을 이용하여 Y108A, Y108F 그리고 Y108W의 새로운 변이체 3개를 얻었다. 대장균을 이용한 대량발현과 affinity column에 의해 정제된 변이체 효소에 대하여 GSH와 CDNB, DCNB, ETA, EPNP 또는 steroid와의 포합반응 활성을 측정하여 야생형과의 활성비교를 하였다. 야생형과의 활성비교에 있어서 각 변이체들의 변화를 볼 수 있었으며, 특히 입체적 장애를 준 Y108W 변이체의 경우 CDNB에 대한 활성이 다른 변이체에 비해 현저히 감소한 반면 ETA에 대한 활성은 증가하였다. 이러한 결과 Tyr108 잔기는 친전자성 기질 결합부위에 관련되는 잔기로 생각되어진다. In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr108 residue in human GST P 1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH with CDNB, DCNB, ETA, EPNP or steroid. The Y108W mutant slightly increased the conjugating activity toward ethacrynic acid(ETA)., but it decreased in specific activity toward 1-chloro-2,4-nitrobenzene(CDNB). On the other hand, the Y108A and Y108F mutants had negligible effect on the activity toward CDNB, ETA and DCNB of the wild type. These results indicated that Tyr108 in human GST P1-1 may contribute to the binding of electrophilic substrate.
Rice glutathione S-transferase의 정제 및 특성
박희중,공광훈 中央大學校 遺傳工學硏究所 1996 遺傳工學硏究論集 Vol.9 No.1
Rice로부터 DEAE-Sepharose와 GSH- garose 및 Sephadex G-75 column chromatography를 이용하여 GST를 분리정제하였다. 정제된 효소는 분자량이 전기동영상에서는 약 28 kDa, gel chromatography상에서는 약 54kDa인 homodimeric 구조이다. 또한 정제된 효소는 다른 종 GST들에 공통적인 특징인 1-chloro-2,4-dinitrobenzene(CDNB)에 대해 높은 기질특이성을 보였으며, S-hexylglutathione과 benanstatin A 에 대해서도 저해효과를 보였다. 효소의 최적온도는 45℃ 를 나타내었다. Glutathione S-transferase (GST) was purified from rice by DEAE-Sepharose, GSH-agarose and Sephadex G-75 column chromatography. The molecular mass of the purified GST was determined to be 54,000 by gel chromatography and 28,000 by SDS-polyacrylamide gel electrophoresis, indicating a homodimeric structure. The enzyme had high substrate activity of the enzyme toward the substrate CDNB was inhibited by S-hexylglutathione and benanstatin A. The optimun pH of enzyme was 7.0 and the optimum temperature was 45℃ with CDNB as a substrate.
최상숙,박희중,조성희 中央大學校 基礎科學硏究所 1997 基礎科學硏究所 論文集 Vol.11 No.-
A sensitive, reproducible and rapid method for the quantitative determination of preservatives in cosmetics was studied by high performance liquid chromatography. The method was based on the HPLC separation of the components on a Lichrosorb RP-18 column with mobile phase having 70% methanol, with sensitivity having 0.05 AUFS at 214nm. The good determined concentrations of Kathon CG, methylparaben and ethyl paraben were 30μg/ml∼ 60μg/ml, imidazolidiny1 urea, 2-bromo-2-ni- tropropane-1,3-diol, propy1 paraben and buty1 paraben were 120μg/ml∼140μg/ml. The retention time was separated Kathon CG 2.67, imidazolidinyl urea 3.52, 2-bromo-2-nitropropane-1,3-diol 4.49, methyl paraben 5.80, ethyl paraben 7.19, propyl paraben 9.80, butyl paraben 14.47 and fluocortolone as the internal standard 12.02.
PTPase의 작용에 menadione이 미치는 활성 기작
함승욱,박희중,임두현 中央大學校 基礎科學硏究所 1995 基礎科學硏究所 論文集 Vol.9 No.-
Vitamin K₃(menadione) has been shown to exhibit a broad range of antitumor activity in human cells as well as a phosphatase cofactor. It also imposed lower level of toxicity than other cancer chemotherapeutic drugs of quinone structure. Juan and Wu have recently demonstrated that the action of menadione is dose-dependent and cell cycle-specific. In this study, we have examined the effect of menadione in the action of cdc25 PTPase which is important of the control of cell cycle progression by the signal transduction pathway. Our mechanistic analysis with the model reaction has led to a new interpretation of the role of menadione. We also investigated that thiolate addition of cdc25 PTPase to menadione leads to directly to irreversible inhibition by covalent modification.
산포도와 머루중의 Polyphenol Oxidase에 관한 연구¹
성찬기,박희중,임흥빈,조성희 中央大學校 基礎科學硏究所 1997 基礎科學硏究所 論文集 Vol.11 No.-
Polyphenol oxidase was partially purified from fresh vitis flexuosa and vitis amurensis and its characteristics were studied. the experimental results were summarized as follows. 1.The enzyme activities were increased during the fruit developement gradually in two sample. 2.The optimum pH and temperature for activity were 5.5 and 30℃, respectively. 3.The enzyme was active with o-phenols and trihydroxyphenol, but inactive toward m-diphenols and monophenols. 4.The ?? value of the enzymes was 15.4 mM in vitis flexuosa and 12.5 mM in vitis amurensis with catechol as substrate. 5.Inhibitors sudies indicated that sodium diethyl-dithiocarbamate, L-cysteine, sodium bisulfite, L-ascorbic acid and 2-mercaptoeyhanol were the most potent. 6.The enzyme activity increament by ?? was much better than those by the other metal ions.