MULTIPLE FORMS OF SNAKE VENOM PHOSPHODIESTERASE FROM Agkistrodon halys

염영나,김두식 한국생화학분자생물학회 (구 한국생화학회) 1987 추계학술대회집 Vol.- No.-

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.

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.

동물대체시험법 국내,외 동향

염영나 한국동물실험대체법학회 2010 한국동물실험대체법학회 학술대회집 Vol.2010 No.1

Betulinic acid 등의 세포독성 조절 연구

염영나,조현영,김현석,황명실,윤은경,이효민,김승희,양지선,양기화 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

과일이나 식물체 등애 천연적으로 존재하면서 돌연변이억제나 항산화효과 등, 암이나 성인병 등에 대한 예방효과를 보이는 것으로 알려진 phytocherilical을 중심으로 aminopept건ase 활성 억제 능력을 조사하여 암전툴과정과 혈관신생과정에서 발된이 증가하는 것으로 알려진 arnjnoepeptidase N APh억제제를 스크리닝하고 암전이 예방이나 암치료에 활용하고자 본 연구를 수행하게 되었다 Leucine andnopeptidase에 대한 식물유래성분(betulinic acid, curcvmin, indomethacin, ellagic acid quercetin, nocodazole, urso:ic acid, resveratrol, h)'pericin, caffeic acid)의 찰성억제능력을 조사하여 그중 촬성억제능력이 탁월한 curcundn, indomethacin, betulinlc 3c펀의 APN enzyme netics를 수헝 하였다. Curcumin의 andnopeptidase 띠 활성을 억제하는 농도와 혈관신생의 억제 농도가 일치하고 APN 단클론항체 처리한 HT1080 세포의 유세포측정결과 curcumin·라 APN 단클론항체가 경정적으로 APN예 결합됨을 확인함에 따라 curcumin이 암전이를 억제하는 기전이 APN 억제에 기인한 것으로 추정되는 결과이다. 한편 Betulinic ac,김는 APN 활성에는 영향을 미치지 못하고, 미토콘드리아의 막전 위를 떤화시키고 caspase근 활성을 증가시켜 apoptosis를 유발하였다. 포한 betulinic acid로 tlibe formation assay와 」'n ufuo CAM assay를 수행한 결과 혈관신생과정을 억제하는 것이 확인되었다 따라저 Betulinic 3fid와 curcumin은 서로 작용기전은 다르지만, 암전이 떼방이나 암치료에 활용가치가 높은 약물로 인식되었다. This study is to evaluate phytochemicals that have inhibitory activity on aminopeptidase N (APN) which is transmembrane metalloprotease to play functional role in matrix degradation and invasion by tumor cells. At first, we did screening of APN inhibitors among the phytochemicals with chemopreventive effect including betulinic acid, curcumin, indomethacin, ellagic acid, quercetin, nocadazole, ursolic acid, reveratrol, hypericin, and caffeic acid etc. by the fluorometric assay using L-alanine 4-methylcoumaryl-7-amide(Ala-AMC) as a substrate. Curcumin has inhibitory effect on APN activity as well as angiogenesis in a same condition(IC_(50) value=about 10μM). Curcumin on APN activity works as a noncompeptitive ingibitor. FCM analysis shows the competitive binding of curcumin and monoclonal antibody of APN to HT1080 cell. This results means that curcumin blocks the angiogenesis by the mechanism of APN inhibition. Betulinic acid which is working as a inhibitor of Leu aminopeptidase, dose not inhibit the aminopeptidase N activitiy. But betulinic acid blocks angiogenesis from the results of tube formation assay and in vivo CAM assay. The angiogenesis blocking mechanism of betulinic acid is supposed to induce apoptosis. These results suggest that curcumin and betulinic acid can be used as angiogenesis blocker and chemopreventive agent of cancer despite of different mechanism.

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 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.

동물대체시험법의 국내,외 동향

염영나 한국동물실험대체법학회 2009 한국동물실험대체법학회 학술대회집 Vol.2009 No.2

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 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.