Deoxynucleoside Kinases from Beef Liver Mitochondria : Characterization of Deoxycytidine and Deoxythymidine Kinase Activities

박인식,Park, In-Shik 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3

dCyd-Sepharose 친화크로마토그래피 칼럼을 이용하여 소의 간 미토콘드리아에 존재하는 데옥시시티딘 키나아제 및 데옥시티미딘 키나아제의 특성을 조사하였다. 소의 간 미토콘드리아 조효소액을 dCyd-Sepharose 칼럼에서 정제시켰을 경우, 데옥시시티딘 키나아제 및 데옥시티미딘 키나아제의 활성이 같은 비율로 dCyd-Sepharose 칼럼에 결합했으며, 아울러 효소 활성은 모두 1 mM 데옥시시티딘 혹은 1 mM 데옥시티미딘을 평형완충용액에 첨가함으로써 용출되었으며, 또한 효소의 용리패턴이 같았다. 그리고 데옥시시티딘 키나아제 및 데옥시티미딘 키나아제의 활성은 전기영동의 결과 같은 $R_f$치를 보였고, Sephacryl S-200의 젤 여과에서도 같은 위치에서 활성의 peak를 나타내었다. Deoxycytidine (dCyd)-Sepharose affinity chromatography was synthesized and utilized to purify both dCyd and deoxythymidine (dThd) kinase activities in beef liver mitochondria. dThd kinase activity was retained on dCyd-Sepharose affinity column and it was eluted by addition of 1 mM dCyd to the equilibration buffer. Moreover, both dCyd and dThd kinase activities were bound to the column and released upon addition of 1 mM dThd to the washing buffer, with relatively similar percentage of recovery. In addition, both dCyd and dThd kinase activities were migrated together in 7% non-denaturating polyacrylamide gel electrophoresis, and were eluted together in gel filtration on Sephacryl S-200.

Glucose oxidase - 한국산 무우 Peroxidase 에 의한 포도당 정량법에 미치는 화원제의 저해효과

박인식,남인,고선옥 ( In Shik Park,In Nam,Sun Ok Kho ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

Control of Deoxyguanosine Kinase from Bovine Liver Mitochondria by Nucleoside Triphosphate

박인식,Park, In-Shik 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3

소 간 mitochondrial deoxyguanosine kinase 활성은 purine 및 pyrimidine nucleoside triphosphate에 의하여 조절되었다. 생리적인 pH(7.4)에서 효소는 deoxythymidine triphosphate와 uridine triphosphate와 같은 pyrimidine triphosphate를 purine nucleoside triphosphate 보다 더욱 기질로 잘 이용하였다. 효소반응의 distal end product인 deoxyguanosine triphosphate와 deoxyinosine triphosphate는 인산공여체로 전혀 작용하지 않았다. Pyrimidine nucleoside triphosphate는 pH7.4에서 효소활성을 증가시켰으나 효소반응의 최적 pH인 5.5에서는 효소활성을 저해하였다. Nucleoside mono- 및 diphosphate에서는 deoxyguanosine kinase 작용의 생성물인 deoxyguanosine monophosphate, deoxyinosine monophosphate, deoxythymidine diphosphate 및 uridine diphosphate가 효소활성을 저해하였다. Nucleoside triphosphate의 경우에는 deoxyinosine triphosphate 및 deoxyguanosine triphosphate가 효소활성을 강하게 저해했으나, guanosme triphosphate와 deoxyadenosine triphosphate는 약하게 저해하였다. Uridine triphosphate와 deoxythymidine triphosphate는 효소활성을 증가하였다. Pyrimidine triphosphate에 의한 효소활성의 증가는 deoxyguanosine triphosphate의 첨가에 의하여 상쇄되었다. The enzymatic activity of mitochondrial deoxyguanosine (dGuo) kinase from bovine liver was controlled by purine and pyrimidine nucleoside triphosphates. At physiological pH (7.4), the enzyme utilized pyrimidine nucleoside triphosphates such as deoxythymidine triphosphate (dTTP) and uridine triphosphate (UTP) better than purine nucleoside triphosphates. The distal end products of the enzyme reaction, deoxyguanosine triphosphate (dGTP) and deoxyinosine triphosphate (dITP), showed no activity as phosphate donors. Pyrimidine nucleoside triphosphates such as dTTP and UTP increased the enzyme activity at pH 7.4, but decreased it at optimum pH (5.5) of the reaction. Among nucleotides mono-and diphosphates tested, only products of the dGuo kinase reaction, deoxyguanosine monophosphate (dGMP), deoxyinosine monophosphate (dIMP), deoxythymidine diphosphate (dTDP) and uridine diphosphate (UDP) caused inhibition of the enzyme. In the case of nucleoside triphosphates, dGTP and dITP produced the strongest inhibition. Purine nucleotides such as guanosine triphosphate (GTP) and deoxyadenosine triphosphate (dATP) also caused considerable inhibition. On the other hand, pyrimidine nucleotides (dTTP, UTP) apparantly increased the enzyme activity. Activation by pyrimidine nucleoside triphosphate can be reversed by the addition of dGTP, and, conversely, inhibition by dGTP can be overcome by addition of dTTP.

Dithiothreitol 에 의한 한국산 무우 Peroxidase 의 비활성화

박인식,서경순 ( In Shik Park,kyung Soon Suh ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

Korean radish peroxidase was inactivated by addition of cystine-specific reagents such as 2-mercaptoethanol or dithiothreitol (DTT) known as Cleland`s reagent. The inactivation of the enzyme by dithiothreitol was affected by concentration of the reagent, temperature of the reaction, and time of incubation, pH of buffer used, and presence of protecting compounds. The inactivation of the enzyme by dithiothreitol was protected by adding substrates, hydrogen peroxide or aminoantipyrine/phenol. Therefore, the inactivation of the enzyme by Cleland`s reagent seems due to the breakage of disulfide bonds in the enzyme.

Korean-Radish Peroxidase for Enzymatic Determination of Glucose

박인식,고선옥,남인,Park, In-Shik,Kho, Sun-Ok,Nam, In 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4

Korean-radish peroxidase can replace horseradish peroxidase for enzymatic determination of glucose. The glucose could be quantitatively assayed by using glucose oxidase and Korean-radish peroxidase. The optimum temperature and pH for glucose assay were of $40^{\circ}C$ and pH 5.5, respectively. Various cations and anions such as $Ag^+$, $Cu^{++}$, $Pb^{++}$, $Fe^{++}$, ${N_3}^-$, ${S_2O_3}^=$, ${S_2O_4}^=$ and ${NO_2}^-$ inhibited the glucose assay completely. The relationship between absorbance and glucose concentration was linear up to $50{\mu}mol$ of glucose under the optimum assay conditions. 한국산 무우 peroxidase는 포도당의 효소적 분석에 주로 사용되는 horseradish peroxidase를 대체할 수 있었다. 포도당은 glucose oxidase- 한국산 무우 peroxidase에 의하여 정량적으로 측정되었다. 포도당 측정의 최적조건은 $40^{\circ}C$ 및 pH 5.5이었다. 양이온 중에서 $Ag^+$, $Cu^{++}$, $Pb^{++}$, $Fe^{++}$ 그리고 음이온 중에서는 ${N_3}^-$, ${SO_3}^=$, ${S_2O_3}^=$, ${S_2O_4}^=$, ${NO_2}^-$는 효소반응을 완전히 저하시킴으로서 한국산 무우 peroxidase를 사용하여 포도당을 측정하는 것을 완전히 방해하였다. 흡광도와 포도당 농도와의 상관관계는 최적조건에서 분석하고자 하는 포도당의 양이 $50{\mu}mol$ 이하에서는 직선관계를 나타내었다.

한국산 무우 peroxidase 에 의한 포도당의 효소적 분석

박인식,고선옥,남인 ( In Shik Park,Sun Ok Kho,In Nam ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4

Korean-radish peroxidase can replace horseradish peroxidase for enzymatic determination of glucose. The glucose could be quantitatively assayed by using glucose oxidase and Korean-radish peroxidase. The optimum temperature and pH for glucose assay were of 40℃ and pH 5.5, respectively. Various cations and anions such as Ag^+, Cu^(++), Pb^(++), Fe^(++), N₃^-, S₂O₃^-, S₂O₄^- and NO₂^- inhibited the glucose assay completely. The relationship between absorbance and glucose concentration was linear up to 50μ㏖ of glucose under the optimum assay conditions.

Inactivation of Korean Radish Peroxidase by Dithiothreitol

박인식,서경순,Park, In-Shik,Suh, Kyung-Soon 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

한국산 무우에서 분리한 peroxidase는 cystine을 선택적으로 환원시키는 2-mercaptoethanol 혹은 dithiothreitonol에 의하여 비활성화 되었다. Dithiothreitol에 의한 효소의 비활성화는 시약의 농도, 반응온도, 반응시간, pH 및 방어물질의 존재에 의하여 영향을 받았다. 그리고 dithiothreitol에 의한 효소의 비활성화는 기질인 hydrogen peroxide 혹은 aminoantipyrine/phenol의 첨가에 의하여 방어되었다. 따라서 dithiothreitol에 의한 효소의 비활성화는 효소중의 disulfide 결합의 손상에 의한 것으로 보인다. Korean radish peroxidase was inactivated by addition of cystine-specific reagents such as 2-mercaptoethanol or dithiothreitol (DTT) known as Cleland's reagent. The inactivation of the enzyme by dithiothreitol was affected by concentration of the reagent, temperature of the reaction, and time of incubation, pH of buffer used, and presence of protecting compounds. The inactivation of the enzyme by dithiothreitol was protected by adding substrates, hydrogen peroxide or aminoantipyrine/phenol. Therefore, the inactivation of the enzyme by Cleland's reagent seems due to the breakage of disulfide bonds in the enzyme.

선택적 LPE방법에 의한 GaAs가판 상의 InP이종접합 박막의 성장

이병택,안주헌,김동근,안병찬,남산,조경익,박인식,장성주,Lee, Byung-Teak,An, Ju-Heon,Kim, Dong-Keun,Ahn, Byung-Chan,Nahm, Sahn,Cho, Kyoung-Ik,Park, In-Shik,Jang, Seong-Joo 한국재료학회 1994 한국재료학회지 Vol.4 No.6

Heteroepitaxial InP/GaAs layers were grown using the selective liquid phase epitaxy (SLPE) technique. It was observed that the optimum LPE conditions were $660^{\circ}C$ growth temperature, $5^{\circ}C$ supercooling, and $0.4^{\circ}C$/min cooling rate. Maximum expitaxial layer overgrowth (ELO) of 110-160$\mu \textrm{m}$ was obtained when the seed was aligned along (112) orientation. Initial melt-back of the substrate was observed but limited to the seed region so that flat In-Ga-As-P layers were grpwn throughout the GaAs substrates. The InP/GaAs heteroepitaxial structure could be obtained by growing an additional InP layer on top of the In-Ga-As-P layer. 선택적 LPE방법을 이용하여 (111)B GaAs 기판 상에 InP연속 박막을 성장하고 그 특성을 평가하였다. 적정 LPE성장조건으로 성장온도 $660^{\circ}C$, 과냉도 $5^{\circ}C$, 냉각속도 $0.4^{\circ}C$/min였으며, 연구된 온도 범위에서 성장온도가 증가할수록 표면형상이 개선되었고 ELO의 넓이가 증가하였다. Seed방향이 <112>방향에서 110-160$\mu \textrm{m}$ 정도의 최대 ELO 넓이가 얻어졌으며 60-80$\mu \textrm{m}$정도의 마스크 간격에서 연속박막을 용이하게 성장할 수 있었다. LPE 성장초기에 기판 용해 현상이 발생하였으며 이에 따라 성장박막의 조성이 대략 $In_{0.85}Ga_{0.15}$As$_{0.01}P{0.99}$으로 변화하고 InP/GaAs계면 및 박막 표면형상이 거칠어졌으나 기판의 성장 부위가 제한됨에 따라 통상적인 LPE박막에 비교하여 매우 개선된 표면형상을 얻을 수 있었다. 두개의 성장융액을 이용하여 1차 박막성장 후 다시 InP 박막을 성장하는 2단성장 방법을 사용하여 순수한 InP/GaAs박막을 성장할 수 있었으며 단면 TEM분석 결과 SLPE성장박막으로 전파하는 활주전위는 산화막 마스크에 의해 효과적으로 차단됨을 알 수 있었다.

소의 간 미토콘드리아에서 존재하는 데옥시뉴클레오시드 키나아제 데옥시시티딘 키나아제 및 데옥시티미딘 키나아제의 특성

박인식 ( In Shik Park ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

Deoxycytidine (dCyd)-Sepharose affinity chromatography was synthesized and utilized to purify both dCyd and deoxythymidine (dThd) kinase activities in beef liver mitochondria. dThd kinase activity was retained on dCyd-Sepharose affinity column and it was eluted by addition of 1 mM dCyd to the equilibration buffer. Moreover, both dCyd and dThd kinase activities were bound to the column and released upon addition of 1 mM dThd to the washing buffer, with relatively similar percentage of recovery. In addition, both dCyd and dThd kinase activities were migrated together in 7% non-denaturating polyacrylamide gel electrophoresis, and were eluted together in gel filtration on Sephacryl S-200.

유근피(楡根皮) 약침의 $NF-{\kappa}B$ 활성 억제능이 생쥐의 Type II Collagen 유발 관절염에 미치는 영향

이아람,변혁,박인식,정찬영,강민주,김은정,이승덕,김갑성,Lee, A-Ram,Byun, Hyuk,Park, In-Shik,Jung, Chan-Yung,Kang, Min-Joo,Kim, Eun-Jung,Lee, Seung-Deok,Kim, Kap-Sung 대한침구의학회 2007 대한침구의학회지 Vol.29 No.2

Objectives : The purpose of this study is to investigate the effectiveness of Ulmus davidiana Planch herbal acupuncture(UA) to inhibit nuclear $factor(NF)-{\kappa}B$ activation on type II collagen-induced arthritis (CIA) in mice. Methods : Using an in vitro test, the synoviocytes picked out from the experimental CIA mice were subcultured. The synoviocyte cells were treated with phorbol-12-myristate-13-acetate(PMA) for 1 hour prior to the addition of indicated concentrations($0.4\;-\;1.0mg/m{\ell}$) of UA, and the cells were further incubated for 24 hours. The in vivo test, $NF-{\kappa}B$ p65, inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2), vascular cell adhesion molecule(VCAM)-1 production and apoptosis was observed by immunohistochemical staining. Results : The PMA-induced $I{\kappa}B$ kinase(IKK), iNOS and COX-2 mRNA expression were dose-dependently decreased in UA treated synoviocytes. Using the in vivo test, the number of eosinophils in mice treated with UA noticeably decreased in the the CIA group(P<0.05 using student t test). In mice treated with UA, there was less cartilage erosion. less bone destruction, mild synovial hyperplasia, mild fibrosis, and mild angiogenesis with less MIP-2 production. By immunohistochemical staining, suppression of $NF-{\kappa}B$ p65, iNOS production, inhibition of COX-2 production, inhibition of VCAM-1 production and inducing apoptosis were observed. Conclusions : These results suggest that UA might be applicable to the therapy of RA to suppress $NF-{\kappa}B$ activation.