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Polynucleotide Kinase 반응에서의 인산화된 효소 중간제 확인
노규승,조영동 ( Gue Seung No,Young Dong Cho ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.3
With the purified polynucleotide kinase from cloned cells in our laboratory, the enzyme reaction was analyzed using [-^(32)P]ATP and [-^(32)P]ATP. Through ADP coupling assay and polyethyleneimime-impregnated cellulose thin layer chromatography, we recognized the fact that the enzyme could generate ADP from ATP in the absence of phosphoryl group acceptor, 5`-OH poly[A]. Based on this result, it was identified that the enzyme was phosphorylated covalently in the presence of ATP and Mg^(2+) by autoradiography following SDS-polyacrylamide gel electrophoresis. Also the isolation of phosphoryl-enzyme which is regarded as intermediate was performed by Sephadex G100 column chromatography. It was measured that the enzyme and phosphoryl group reacted by 1:1 ratio and the bound phosphoryl group was more stable in acid than in alkali.
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Identification of Phosphoryl-enzyme Intermediate in Polynucleotide kinase Reaction
노규승,조영동,No, Gue-Seung,Cho, Young-Dong Korean Society for Biochemistry and Molecular Biol 1990 한국생화학회지 Vol.23 No.3
With the purified polynucleotide kinase from cloned cells in our laboratory, the enzyme reaction was analyzed using $[-^{32}P]$ATP and $[-^{32}P]$ATP. Through ADP coupling assay and polyethyleneimime-impregnated cellulose thin layer chromatography, we recognized the fact that the enzyme could generate ADP from ATP in the absence of phosphoryl group acceptor, 5'-OH poly[A]. Based on this result, it was identified that the enzyme was phosphorylated covalently in the presence of ATP and $Mg^{2+}$ by autoradiography following SDS-polyacrylamide gel electrophoresis. Also the isolation of phosphoryl-enzyme which is regarded as intermediate was performed by Sephadex G100 column chromatography. It was measured that the enzyme and phosphoryl group reacted by 1:1 ratio and the bound phosphoryl group was more stable in acid than in alkali. 본 실험실에서 클로닝된 균주로부터 순수하게 정제된 polynucleotide kinase의 효소반응을 방사성 동위원소로 표지된 $[{\alpha}-^{32}P]$ATP, $[{\gamma}-^{32}P]$ATP를 사용하여 분석하였다. ADP coupling assay와 Polyethyleneimine-impregnated cellulose thin layer 크로마토그래피를 통하여, Polynucleotide kinase는 기질인 5'-OH poly[A]가 없는 상태에서 ATP로부터 ADP를 형성한다는 사실을 발견하였다. 이러한 사실을 토대로 이 효소는 ATP와 $Mg^{2+}$가 존재하는 조건하에서 공유결함으로 인산화된다는 것을 sodium dodecyl sulfate-polyacrylamide gel electrophoresis 후 autoradiography하는 방법을 통하여 확인할 수 있었다. 또한 Sephadex G-100컬럼 크로마토그래피에 의해 인산화된 효소 중간체를 분리하였다. 효소와 인산기는 1:1 비율로 반응하는 것으로 나타났으며 공유결합된 인산기는 산성보다는 알칼리성에서 안정하였다.
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T4 Polynucleotide Kinase Gene Cloning by ${\lambda}$gt 11 Vector
김주훈,권세창,노규승,조영동,Kim, Joo-Hoon,Geun, Se-Chang,No, Gue-Seung,Cho, Young-Dong Korean Society for Biochemistry and Molecular Biol 1989 한국생화학회지 Vol.22 No.1
${\lambda}$gt 11 vector를 이용하여 유전자 조작에 유용하게 사용되는 polynucleotide kinase의 클로닝을 시도하였다. T4 DNA를 EcoRI으로 잘라 polynucleotide kinase 유전자를 얻었으며 ligase로 유전자를${\lambda}$gt 11 vector와 연결시켜 in vitro packaging 시킨 다음 E. coli Y 1090에 감염시켰다. Plaque hybridization, Eco RI-SmaI 제한효소 절단에 의하여 polynucleotide kinase 유전자가 클로닝된 phage를 선별하고 DE-81 paper binding assay, silica gel thin layer chromatography 등을 이용하여 이 phage로부터 단백질 수준에서 polynucleotide kinase를 생산하는 균주를 선발하였다. Polynucleotide kinase를 생산하는 것이 확인된 phage를 E.coli Y 1089에 감염시켜 lysogenic bacteria를 만든 다음 Western blot 방법으로 polynucleotide kinase가 생산되는 것을 재확인하였다. 클로닝한 균주에서 생산된 polynucleotide kinase는 gel filtration상에서 분자량이 66,000 dalton이고 sodium dodesyl sulfate polyacrylarnide gel electrophoresis상에서 분자량이 33,000 dalton으로 나타나 dimer이며 ${\beta}$-galactosidase 분자의 일부와 fusion된 polynucleotide kinase는 만들어지지 않았다. 클로닝하여 얻은 polynucleotide kinase는 T4에서 얻은 polynucleotide kinase와 chromato graphy에서 용출되는 성질과 5'-kinase specific activity는 비슷하였다. 그러나 3'-phosphatase의 specific activity는 T4에서 얻은 polynucleotide kinase의 1/5 정도밖에 되지 않았다. 클로닝한 균주에서 얻은 polynucleotide kinase의 양은 T4를 감염시켜 얻을 때보다 7~8배 더 많이 얻을 수 있었다. Attempts were made to clone polynucleotide kinase widely used in gene manipulation using ${\lambda}$gt11 vector. Polynucleotide kinase gene obtained from T4 DNA by EcoRI digestion was ligated to ${\lambda}$gt11 vector and in vitro packaged, then used to infect E. coli Y1090. The selection of phage strain producing polynucleotide kinase from the cloned phages was done using plaque hybridization, EcoRI-SmaI double digestion in DNA level and DE-81 paper binding assay, silica gel thin layer chromatography and autoradiography in protein level. E. coli Y1089 was infected with the cloned phage producing polynucleotide kinase in order to get lysogenic bacteria. Molecular weight measured by sodium dodesyl sulfate polyacrylamidegel electrophoresis and native molecular weight by gel filtration were 33,000 and 66,000 dalton, respectively, indicating a dimeric enzyme which was turned out to be unfused with the moiety of the ${\beta}$-galactosidase molecule. Chromatographic behaviors and 5'-kinase specific activity of polynucleotide kinase from T4 and that of the cloned lysogenic bacteria were similar. But specific activity of 3'-phosphatase from the cloned lysogenic bacteria was 1/5 of that from T4. The amount of polynucleotide kinase from the lysogenic bacteria was 7-8 fold compared with that from T4.
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