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.

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.

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.

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

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.

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.

Nucleoside Triphosphate 에 의한 소 간 Deoxyguanosine Kinase 활성의 조절

박인식 ( In Shik Park ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3

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.

Aspergillus sp . 가 생산하는 Glucoamylase Isozymes 의 성질

박인식(In Shik Park),정영호(Young Ho Chung) 한국미생물생명공학회 1988 한국미생물·생명공학회지 Vol.16 No.4