The Incorporation of Eucaryotic Gene Using Ti Plasmid Vector: I. The Introduction of Yeast Homoserine Dehydrogenase Gene into Agrobacterium tumefaciens

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Sun-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

효모에 있어서 threonine과 methionine 생합성에 관여하는 효소들 중 하나인 homoserine dehydrogenase가 식물체내로 도입된 후, 그 역할을 조사하고자 Ti plasmid에서 유도된 pGA658 vector와 homoserine dehydrogenase 유전자 함유 DNA의 재조합을 시도하였다. 그 결과 pGA658 vector의 nos promoter 다음에 효모의 homoserine dehydrogenase 유전자를 함유한 DNA 절편이 방향이 반대로 삽입된 재조합 plasmids가 성공적으로 만들어졌다. 형질전환된 E. coli와 Agrovbacterium tumefaciens는 적절한 항생물질 함유배지에서 저항성을 나타내었으며, 그들의 plasmid를 분리하여 여러가지 제한효소를 이용한 크기를 조사해 보니, 예상되는 크기와 일치하는 것을 관찰할 수 있었다. 또한 형질전환된 E. coli와 A. tumefaciens내에서의 yeast homoserine dehydrogenase 유전자 발현을 조사하기 위해 그 효소의 활성을 조사해 본 결과, 형질전환된 E. coli에서는 대조군에 비해 약간의 증가를 나타내었으나, 형질전환된 A. tumefaciens에서는 변화가 없었다. DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli, but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

소의 간 미토콘드리아 Matrix Aldehyde Dehydrogenase의 반응메카니즘에 관한 연구

김선진,주충노,Kim, Sun-Jin,Joo, Chung-No 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5

소의 간 mitochondria의 matrix에서 분리 정제한 aldehyde dehydrogenase(EC 1.2.1.3)의 초기반응속도와 NADH에 의한 반응억제 연구로부터 이 효소반응은 sequential mechanism이며 $NAD^+$가 효소에 먼저 결합한 후 aldehyde가 결합하고 생성물인 산이 먼저 이탈된 후 NADH가 이탈되는 ordered mechanism임을 알 수 있었다. 또한 이 효소는 dehydrogenase 활성 이외에 높은 esterase 활성을 나타내었다. Chloral hydrate에 의한 이 ALDH의 dehydrogenase 활성과 esterase 활성의 저해양상을 조사한 결과 $NAD^+$ 존재하에서는 dehydrogenase 활성을 경쟁적으로 저해하였지만 esterase 활성에 대해서는 mixed type의 저해양상을 나타내었다. Disulfiram의 억제반응에서 dehydrogenase의 반응 기질인 propionaldehyde는 disulfiram에 의한 dehydrogenase 활성저해를 보호하였지만 esterase 활성의 저해는 보호하지 못하였고 esterase의 반응기질인 p-nitrophenylacetate도 esterase의 활성저해를 보호하였지만 disulfiram에 의한 dehydrogenase 활성의 저해를 보호하지 못하였다. 또한 $100{\mu}M$ 이상의 $NAD^+$ 존재하에서는 esterase 반응이 $NAD^+$ 자체에 의해 저해되는 것으로 보아 dehydrogenase 반응과 esterase 반응이 반드시 같은 부위에서 일어난다고 생각하기는 어렵다. The initial velocity data and NADH inhibition behavior of the purified bovine liver mitochondrial matrix aldehyde dehydrogenase (EC 1.2.l.3, ALDH) indicated that the ALDH reaction might be proceeded by sequential ordered mechanism, first $NAD^+$ is bound with the enzyme followed by aldehyde and then the oxidized product (acid) would be removed followed by NADH This enzyme also showed esterase activity. We examined its esterase activity as well as dehydrogenase activity through various kinetic approchs and chemical modification techniques and we found that the active site of dehydrogenase reaction and esterase reaction might be different.

소의 간 미토콘드리아 Matrix Aldehyde Dehydrogenase의 정제 및 특성에 관한 연구

김선진,주충노,Kim, Sun-Jin,Joo, Chung-No 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5

소의 간 미토콘드리아의 matrix 분획으로부터 aldehyde dehydrogenase(EC 1.2.1.3, ALDH)를 황산암모늄 분별침전, DEAE-Sephacel, hydroxyapatite, blue-Sepharose CL-6B column chromatography로 정제하였다. S-300 gel filtration 방법으로 측정한 이 효소의 분자량은 230,000 dalton이고, 이 효소의 subunit을 SDS-polyacrylamide gel 전기이동법으로 조사한 결과 동일한 subunit(분자량 : 57,400 dalton)으로 구성된 homotetramer임을 알 수 있었다. 이 효소의 최적 pH는 9.5였고, $25{\sim}37^{\circ}C$에서 매우 안정하였다. Acetaldehyde와 propionaldehyde에 대한 $K_m$값은 각각 $9.6{\mu}M$, $6.5{\mu}M$이었으나 succinic semialdehyde나 indole-3-acetaldehyde에 대한 반응성은 매우 낮았고, 방향족 aldehyde에 대해서는 전혀 반응을 하지 않았다. 따라서 이 효소는 주로 short chain aliphatic aldehyde의 대사에 관여하는 것으로 생각된다. Bovine liver mitochodrial matrix aldehyde dehydrogenase (ALDH) was purified by ammonium sulfate precipitation, DEAE-Sephacel, hydroxyapatite and blue-Sepharose CL-6B column chromatography. The molecular weight of this enzyme was determined to be 230 KDa by S-300 gel filtration and it was realized that this enzyme was consisted of four identical subunits and molecular weight of its subunit was found to be 57.4 KDa by SDS-polyacrylamide gel electrophoresis, indicating that this enzyme is homotetramer. This ALDH was stable at $25{\sim}37^{\circ}C$ and its optimum pH was 9.5. The $K_m$ values of this enzyme for acetaldehyde and propionaldehyde were $9.6{\mu}M$ and $6.5{\mu}M$ respectively but $K_m$ values for biogenic aldehydes such as succinic semialdehyde and indole-3-acetaldehyde were relatively high ($10^{-4}$ mM level), but not reactive with aromatic aldehydes, suggesting that this enzyme might participate mainly in the oxidation of short chain aliphatic aldehydes.

Ti plasmid vector 를 이용한 진핵세포 유전자의 도입에 관한 연구

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Sun Joo Hong,Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1

DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli. but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

소의 간 미토콘드리아 Matrix Aldehyde Dehydrogenase 의 반응메카니즘에 관한 연구

김선진,주충노 ( Sun Jin Kim,Chung No Joo ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5

The initial velocity data and NADH inhibition behavior of the purified bovine liver mitochondrial matrix aldehyde dehydrogenase (EC 1.2.1.3, ALDH) indicated that the ALDH reaction might be proceeded by sequential ordered mechanism, first NAD^+ is bound with the enzyme followed by aldehyde and then the oxidized product (acid) would be removed followed by NADH. This enzyme also showed esterase activity. We examined its esterase activity as well as dehydrogenase activity through various kinetic approchs and chemical modification techniques and we found that the active site of dehydrogenase reaction and esterase reaction might be different.

소의 간 미토콘드리아 Matrix Aldehyde Dehydrognease 의 정제 및 특성에 관한 연구

김선진,주충노 ( Sun Jin Kim,Chung No Joo ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5

Bovine liver mitochodrial matrix aldehyde dehydrogenase (ALDH) was purified by ammonium sulfate precipitation, DEAF-Sephacel, hydroxyapatite and blue-Sepharose CL-6B column chromatography. The molecular weight of this enzyme was determined to be 230 KDa by S-300 gel filtration and it was realized that this enzyme was consisted of four identical subunits and molecular weight of its subunit was found to be 57.4 KDa by SDS-polyacrylamide gel electrophoresis, indicating that this enzyme is homotetramer. This ALDH was stable at 25∼37℃ and its optimum pH was 9.5. The K_m values of this enzyme for acetaldehyde and propionaldehyde were 9.6 μM and 6.5μM respectively but K_m values for biogenic aldehydes such as succinic semialdehyde and indole-3-acetaldehyde were relatively high (10^(-4) mM level), but not reactive with aromatic aldehydes, suggesting that this enzyme might participate mainly in the oxidation of short chain aliphatic aldehydes.