한국산 뱀독의 Phosphodiesterase 정제와 특성

염영나,최혜림,김두식 ( Young Na Yum,Hye Lim Choi,Doo Sik Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.1

Phosphodiesterase (EC 3.1.4.1) has been purified to homogeneity from the venom of Korean snake Agkistrodon halys. A combination of ion-exchange, gel filtration and affinity chromatographic procedure leading to 3,600 fold purification of the phosphodiesterase with specific activity of 81.2 units/㎎ was established. Molecular weight of the purified enzyme, a monomeric glycoprotein, was identified to be 110,000. Apparent K_m, and V_(max) values for the hydrolysis of bis-p-nitrophenyl phosphate (bis-PNP) were determined to be 2.44 mM and 4.5×10^(-4) moles/1·min. respectively. Activation energy of the enzyme catalysis toward the bis-PNP hydrolysis was estimated to be 10.4 KJ/㏖. The enzyme exhibited its maximum catalytic activity at pH 9.5. Both exonuclease and endonuclease activities of the purified enzyme were demonstrated using plasmid DNA as a substrate. It is clear that the phosphodiesterase is a bifunctional enzyme which retains intrinsic endonuclease function as well as exonuclease activity in a single protein molecule.

뱀독 Phosphodiesterase 활성부위의 화학변형

염영나,김두식 ( Young Na Yum,Doo Sik Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Snake venom phosphodiesterase is inactivated by nucleotide analogue 5`-fluorosulfonylbenzoyladenosine. Exonucleolytic activity of the enzyme toward bis-p-nitrophenylphosphate is inhibited with a time-dependent and irreversible manner following pseudo-first-order kinetics. The apparent K₁ and kinact were determined to be 0.16 mM and 0.32 min^(-1), respectively. At the same time, the endonucleolytic function toward plasmid DNA, which was analyzed by agarose gel electrophoresis and by fluorometric method using ethidium bromide, was also inactivated. Kinetic data suggest one mole of the affinity labeling nucleotide was bound per mol of the inactivated enzyme. It appears that the catalytic site responsible for both of the exonuclease and endonuclease activities is located in a single functional domain of the monomeric enzyme molecule.

뱀독 Phsphodiesterase 의 Endonuclease 활성에 대한 기질특이성

염영나,김두식 ( Young Na Yum,Doo Sik Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Endonucleolytic cleavage sites of pUC19 by snake venom phosphodiesterase were identified. Limited digestion of supercoiled pUC19 DNA by the phosphodiesterase yields unit length linear form of the plasmid plus two discrete fragments which are 1579 by and 1107 bp. Studies of restriction enzyme mapping analysis indicate that the cleavage sites are 1398 and 2505 of pUC19. The cleavage site located at 1398 which is known as a cruciform structure, coincides with the position recognized by single-strand specific nuclease S1. The 2505 region appears to form a hairpin structure with (A+T)-rich sequence. It is concluded that the endonuclease activity of snake venom phophodiesterase recognizes certain specific secondary structures such as cruciform or (A+T)-rich hairpin region of DNA.

한국산 뱀독의 Phosphodiesterase 정제와 특성

염영나,최혜림,김두식,Yum, Young-Na,Choi, Hye-Lim,Kim, Doo-Sik 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.1

Phosphodiesterase (EC 3.1.4.1) has been purified to homogeneity from the venom of Korean snake Agkistrodon halys. A combination of ion-exchange, gel filtration and affinity chromatographic procedure leading to 3,600 fold purification of the phosphodiesterase with specific activity of 81.2 units/mg was established. Molecular weight of the purified enzyme, a monomeric glycoprotein, was identified to be 110,000. Apparent $K_m$, and $V_{max}$ values for the hydrolysis of bis-p-nitrophenyl phosphate (bis-PNP) were determined to be 2.44 mM and $4.5{\times}10^{-4}moles/l{\cdot}min$. respectively. Activation energy of the enzyme catalysis toward the bis-PNP hydrolysis was estimated to be 10.4 KJ/mol. The enzyme exhibited its maximum catalytic activity at pH 9.5. Both exonuclease and endonuclease activities of the purified enzyme were demonstrated using plasmid DNA as a substrate. It is clear that the phosphodiesterase is a bifunctional enzyme which retains intrinsic endonuclease function as well as exonuclease activity in a single protein molecule. 한국산 살모사(Agkistrodon halys)의 독으로부터 phosphodiesterase(EC 3.1.4.1)를 순수 하게 정제하였다. 5'-AMP 친화성 크로마토그라피를 포함하는 네 단계의 분리과정을 거쳐 3,600배 정제한 phosphodiesterase의 비활성도는 81.2 units/mg이었다. 정제된 효소는 monomer 구조의 당단백질로서 그 분자량이 110,000으로 확인되었다. 이 효소의 $K_m$과 $V_{max}$는 각각 $2.44{\times}10^{-3}M$, $4.5{\times}10^{-4}moles/l{\cdot}min$으로 측정되었고 bis-p-nitrophenyl phosphate의 가수분해를 촉매하는 활성화에너지는 10.4 KJ/mole이며 효소활성에 대한 최적 pH는 9.5로 나타났다. 순수하게 정제된 효소를 이용하여 plasmid DNA를 기질로 사용함으로써 phosphodiesterase의 exonuclease 및 endonuclease 활성을 모두 확인하였다. 따라서 한국산 뱀독의 phosphodiesterase는 exonuclease 활성은 물론 endonuclease 기능도 함께 보유하고 있는 이중성 효소임을 증명하였다.

뱀독 Phosphodiesterase의 이중성 촉매기능

염영나,김두식,Yum, Young-Na,Kim, Doo-Sik 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Exonuclease와 endonuclease 활성을 모두 가지고 있는 뱀독 phosphodiesterase의 촉매 부위에 대한 특성 연구를 수행하였다. 5’-AMP에 의하여 phosphodiesterase의 exonuclease 활성은 경쟁적으로 억제되며, 동시에 endonuclease 활성도 억제됨을 관찰하였다. N-bromosuccinimide 또는 diisopropylfluorophosphate를 이용한 효소의 화학변형 실험결과 역시 두 측매활성이 모두 비가역적2-.로 상실되지만 5’-AMP나 5’-ATP는 이와 같은 화학변형에 대해 현저한 효소활성 보호효과를 나타냈다. 실험적 증거를 토대로 하여 phosphodiesterase의 exo 및 endonuclease 활성은 110K dalton의 monomer 효소단백질 구조에 독립적으로 위치하는 것이 아니라 동일 촉매부위에 존재함을 입증하였다. Dual nuclease activities of snake venom phosphodiesterase from Agkistrodon halys were characterized. Exonucleolytic activity of the enzyme for the hydrolysis of synthetic substrate bis-p-nitrophenyl phosphate is competitively inhibited in the presence of adenosine 5’-mono-phosphate. At the same time, the endonuclease function is also inactivated by the mononuc-leotide. Chemical modification of the purified enzyme by either N-bromosuccinimide or diiso-propylfluorophosphate results in loss of the both catalytic activities. However, adenosine 5'-triphosphate or the corresponding mononucleotide could afford protection against the chemi-cal modification of the enzyme. Several lines of experimental evidence indicate that the catalytic site responsible for both of the exonuclease and endonuclease activities is located in a single functional domain of the monomeric enzyme molecule.

뱀독 Phosphodiesterase의 Endonuclease 활성에 대한 기질특이성

염영나,김두식,Yum, Young-Na,Kim, Du-Sik 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

뱀독 phosphodiesterase의 endonuclease 활성에 대한 pUC19 DNA의 절단부위를 확인하였다. Phosphodiesterase의 endonuclease 반응에 의하여 supercoil 형태의 pUC19은 linear형태 이외에 1579 bp와 1107 bp의 DNA 단편들로 분해된다. 제한효소 mapping 분석방법으로 결정한 pUC19의 절단부위는 1398과 2505였다. Cruciform을 이루고 있는 1398 위치의 절단부위는 single-strand specific nuclease S1의 작용부위와 일치하는 것으로 밝혀졌으며 2505 위치는 hairpin 구조를 이루는 것으로 예상된다. 그러므로 뱀독 phosphodiesterase의 endonuclease 활성은 DNA의 cruciform 또는 (A+T)-rich hairpin 구조를 인식하여 가수분해함을 증명하였다. Endonucleolytic cleavage sites of pUC19 by snake venom phosphodiesterase were identified. Limited digestion of supercoiled pUC19 DNA by the phosphodiesterase yields unit length linear form of the plasmid plus two discrete fragments which are 1579 by and 1107 bp. Studies of restriction enzyme mapping analysis indicate that the cleavage sites are 1398 and 2505 of pUC19. The cleavage site located at 1398 which is known as a cruciform structure, coincides with the position recognized by single-strand specific nuclease S1. The 2505 region appears to form a hairpin structure with (A+T)-rich sequence. It is concluded that the endonuclease activity of snake venom phophodiesterase recognizes certain specific secondary structures such as cruciform or (A+T)-rich hairpin region of DNA.

뱀독 Phosphodiesterase 의 이중성 촉매기능

염영나,김두식 ( Young Na Yum,Doo Sik Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Dual nuclease activities of snake venom phosphodiesterase from Agkistrodon halys were characterized. Exonucleolytic activity of the enzyme for the hydrolysis of synthetic substrate bis-p-nitrophenyl phosphate is competitively inhibited in the presence of adenosine 5`-mono-phosphate. At the same time, the endonuclease function is also inactivated by the mononuc-leotide. Chemical modification of the purified enzyme by either N-bromosuccinimide or diiso-propylfluorophosphate results in loss of the both catalytic activities. However, adenosine 5`-triphosphate or the corresponding mononucleotide could afford protection against the chemi-cal modification of the enzyme. Several lines of experimental evidence indicate that the catalytic site responsible for both of the exonuclease and endonuclease activities is located in a single functional domain of the monomeric enzyme molecule.

KoCVAM’s Activities to Expand and Strengthen National and International Cooperation on Alternative Methods

Young Na Yum(염영나) 한국실험동물학회 2012 한국실험동물학회 학술발표대회 논문집 Vol.2012 No.2

말초혈액을 이용한 초고속 유전독성평가법 개발 연구

안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.

연구논문 : 말초혈액을 이용한 초고속 유전독성평가법 개발 연구

안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.