어피니티 크로마토그라피에 의한 Glucose - 6 - Phosphate Dehydrogenase 의 정제

최양도,변시명,한문희 ( Yang Do Choi,Si Myung Byun,Moon H . Han ) 생화학분자생물학회 1979 BMB Reports Vol.12 No.3

Using the combination procedure of Cibacron Blue F3G-A Sepharose 4B affinity chromatography and hydroxyapatite chromatography, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified rapidly and inexpensively from crude extract of the cell. The Blue-Sepharose affinity chromatography could not separate the two enzymes, glucose-6-phosphate dehydrogenase and lactic dehydrogenase with application of a simple KCl salt gradient. These two enzymes were, however, resolved by the following hydroxyapatite chromatography. The purified enzyme was proved to be homogeneous by polyacrylamide disc gel electrophoresis and SDS gel electrophoresis. The synthesized Blue-Sepharose adsorbent showed dye substitution of 105.94 μmole/g dry gel of 2.27 μmole/㎖ packed gel. The binding capcity of glucose-6-phosphate dehydrogenase was 14.9 U/㎖ packed gel. Blue Dextran 2,000 an analogue of Blue-Sepharose, inhibited the enzyme competitively with respect to NAD^+ and the inhibition constant was determined to be 0.98 μM.

Purification of Glucose-6-Phosphate Dehydrogenase by using Cibacron Blue F3G-A Sepharose 4B Affinity Chromatography

최양도,변시명,한문희,Choi, Yang-Do,Byun, Si-Myung,Han, Moon-H. 생화학분자생물학회 1979 한국생화학회지 Vol.12 No.3

Leuconostoc mesenteroides 에서 추출한 glucose 6-phosphate dehydrogenase를 Cibacron Blue F3GA-Sepharose 4B 어피니티 크로마토그라피와 하이드록시 아파타이트 크로마토그라피 방법에 의해 신속하고 경제적으로 순수 분리하였다. 배양한 세포를 초음파로 파괴하여 얻은 조효소액을 Blue-Sepharose에 어피니티 크로마토그라피한 결과 단순히 KCI gradient만을 사용해서는 glucose-6-phosphate dehydrogenase와 함께 lactate dehydrogenase가 혼합된 부분 정제밖에 얻을 수가 없었다. 그러나 이 두 효소는 다음 단계인 하이드록시 아파타이트 크로마토그라피에 의해 완전히 분리되어 순수한 glucose-6-phosphate dehydrogenase를 얻었다. 이렇게 하여 얻은 glucose-6-phosphate dehydrogenase는 polyacrylamide겔 전기 영동파 SDS겔 전기영동에 의해 그 순수함이 증명되었다. 본 실험을 위해 합성 사용한 Blue-Sepharose 겔의 Cibacron Blue F3G-A 치환도는 건조시킨 겔 1 ml에 대해 $105.94\;{\mu}mole$, 충진된 겔 1 g에 대해 $2.27\;{\mu}mole$을 보여 주었다. 당체가 효소를 결합할 수 있는 능력은 충진 겔 1 ml에 대해 14.9 U이었다. Blue-Sepharose와 유사한 물질인 Blue Dextran 2,000은 효소에 대해 $NAD^+$와 경쟁적 저해 현상을 보였으며 저해상수는 $0.98\;{\mu}M$ 이었다. Using the combination procedure of Cibacron Blue F3G-A Sepharose 4B affinity chromatography and hydroxyapatite chromatography, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified rapidly and inexpensively from crude extract of the cell. The Blue-Sepharose affinity chromatography could not separate the two enzymes, glucose-6-phosphate dehydrogenase and lactic dehydrogenase with application of a simple KCl salt gradient. These two enzymes were, however, resolved by the following hydroxyapatite chromatography. The purified enzyme was proved to be homogeneous by polyacrylamide disc gel electrophoresis and SDS gel electrophoresis. The synthesized Blue-Sepharose adsorbent showed dye substitution of $105.94\;{\mu}mole/g$ dry gel of $2.27\;{\mu}mole/ml$ packed gel. The binding capcity of glucose-6-phosphate dehydrogenase was 14.9 U/ml packed gel. Blue Dextran 2,000 an analogue of Blue-Sepharose, inhibited the enzyme competitively with respect to $NAD^+$ and the inhibition constant was determined to be $0.98\;{\mu}M$.

진핵세포에서의 pre - mRNA Splicing 메카니즘

최양도 ( Yang Do Choi ) 한국생화학분자생물학회 (구 한국생화학회) 1988 생화학총설집 Vol.2 No.-

Bacillus licheniformis Xylanase 의 정제와 특성

최양도(Yang Do Choi),한문희(Moon Hi Han),김진미(Jin Mee Kim) 한국미생물생명공학회 1983 한국미생물·생명공학회지 Vol.11 No.3

Aspergillus niger 의 Hemicellulase 계 효소에 관한 연구 (Ⅰ) D - xylanase 계 효소의 정제와 재조합 -

최양도(Yang Do Choi),이희종(Hee Jong Lee),한문희(Moon H . Han) 한국미생물생명공학회 1983 한국미생물·생명공학회지 Vol.11 No.1

Characteristics of Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides

변시명,최양도,한문희,Byun Si Myung,Yang Do Choi,Moon H. Han Korean Chemical Society 1979 대한화학회지 Vol.23 No.4

저자들은 Cibacron Blue F3G-A Separose 컬럼 어피니티크로마토그래피에 의하여 GIn-cose-6-phosphate dehydrogenase를 Leuconostoc mesenteroides로부터 순수 분리한 바 있다. 이 효소를 사용하여 효소특성을 조사한 결과 분자량은 Sephadex G-200 컬럼에 의해 112,000이었으며 최적온도는 50$^{\circ}$, 활성화에너 지는 8.36kcal/mole 불활성화에너지는 -58.2kcal/mole로 나타났다. $NADP^+$를 조효소로 사용하였을때 최적 pH7.8에서 K_{G6p}:76.9${\mu}$M, ${\alpha}K_{NADP}:\;7.46{\mu}M,\;{\alpha}KNNADP:\;7.l4{\mu}M$이었으며 같은 조건에서 $NAD^+$를 조효소로 사용하였을때 $K_{G6P}:\;53.65{\mu}M,\;K_{NAD}:\;115.2{\mu}M\;{\alpha}K_{NAD}:\;707.2{\mu}M$이었다. 따라서 $NADP^+$ 및 $NAD^+$를 조효소로 사용한 경우에 있어서 ${\alpha}$ 값은 각각 1과 6으로 나타났다. pH변화에 따른 반응속도상수의 변화에 의하면 $NAD^+$를 조효소로 하였을때 최적 pH는 7.8 이었고 pKa가 7.2인 활성기와 ${\mu}Kb$가 9.0∼9.6인 활성기가 효소와 기질의 상호작용에 관여함을 알았다. 이중 pKa 7.2인 활성기를 밝히기 위하여 효소를 광산화와 carboxymethylation을 시킨결과 histidine의 imidazole기임을 알수 있있다. Glucose 6-phosphate dehydrogenase of Leuconostoc mesenteroides which was purifid by an affinity chromatography was studied on the characterization, kinetics and chemical modification. The apparent molecular weight of the enzyme was 112,000 by the gel filtration method of Sephadex G-200 column. The optimum temperature of $NAD^+$-linked reation was 50$^{circ}C$ and the activation energy and the heat of inactivation were 8.36 kcal/mole and -58.2kcal/mole, respectively. The steady state kinetic study showed KG6P, Kemp, and CX KNADP to be 76.9 PM, 7.46${\mu}M$ and 7.14 ${\mu}M$, respectively, and KGGP, KNAD,and aKNm to be 53.7${\mu}M$, 115.2${\mu}M$ and 702.2${\mu}M$ for the $NAD^+$-linked reaction at pH 7.8, optimum pH. The pH dependent kinetic constants suggested that the two ionizing groups whose pKa is 7.2 .and pKb is 9.0-9.6 were involved in the enzyme-substrate interaction. Evidence by photooxidation and carboxymethylation of the enzyme suggested that the imidazole group of histidine with pKa group may participate in the catalytic site.

대두 glyciin subunit A2B1 의 cDNA 유전자 분리

김정호,최양도 ( Chung Ho Kim,Yang Do Choi ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Glycinin is the most abundant storage protein in soybean. A molecule of glycinin is composed of 6 subunits each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one. These polypeptides are synthesized as a precursor which is cleaved to yield A- and B-polypeptides. To study the structure and expression mechanism of the gene, isolation of cDNA clones for soybean glycinin was attempted by immunological screening of a soybean cDNA library with antibodies. The cDNA expression library was constructed with double stranded cDNA of polyadenylated mRNA into λgt11 expression vector. Antibodies to A- and B-polypeptides of glycinin subunit which correspond to N- and C-terminal domain of mRNA translate, respectively, were employed. Eight clones for glycinin, 4 with anti-A and 4 with anti-B polypeptide antibody, were isolated by immunoscreening. The clone λG1A1 with 1186 bp insert was characterized to be a partial cDNA clone for glycinin subunit A₂B_(1a) by nucleotide sequencing analysis and the expression in E. coli. It encodes a half of A₂- and entire B_(la)-polypeptide. The mRNA size of glycinin was demonstrated to be 1.8 kb by Northern blot analysis. It was expressed only in seeds maintaining tissue-specific expression pattern, which was controlled at the level of transcription rather than at translation.

Molecular Cloning of cDNA Encoding the Precursor to the Glycinin $A_2B_{1a}$ Subunit of Soybean

김정호,최양도,Kim, Chung-Ho,Choi, Yang-Do 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

Glycinin은 대두의 주된 저장 단백질이다. Glycinin 분자는 6개의 subunit으로 이루어져 있으며, 각 subunit은 산성 및 염기성 polypeptide로 구성되어 있다. 이들 polypeptide는 전구체로 합성되어진 후 내부 절단에 의해 산성 및 염기성 polypeptide로 갈라진다. Glycinin 유전자의 구조와 발현 메카니즘을 규명하기 위해 cDNA 유전자 발현 은행을 제조한 후, 항체를 사용한 면역 선별법에 의해 cDNA 유전자를 선별하였다. cDNA 유전자 발현 은행은 mRNA를 이용하여 double stranded cDNA를 합성한 후 ${\lambda}$gt11 발현 운반체에 삽입 제조하였다. 항체는 glycinin 산성 및 염기성 polypeptide 각각에 대한 것을 사용했는데, 이들의 항원은 mRNA 에서 합성된 전구체 단백질의 N-terrninal과 C-terminal 에 해당한다. 8개의 cDNA clone을 분리하였는데 4개는 산성 polypeptide에 대한 항체, 4개는 염기성 polypetide에 대한 항체에 의해 선별된 것이다. 염기서열을 결정한 결과, 그 중 cDNA 길이가 1185 bp인${\lambda}$G1A1 clone 은 glycinin $A_2B_{1a}$ subunit을 code하는 cDNA 유전자의 부분 clone임을 알았다. 이 clone은 산성 polypeptide의 절반과 염기성 polypeptide의 전부에 해당하였다. 이 cDNA 유전자에 해당하는 mRNA의 크기는 약 1.8 kb 임을 Northem blot 결과 알 수 있었다. Glycinin 유전자는 대두 종자에서만 발현이 되었으며, 이러한 조직 특이성 발현 형태는 transcription 단계에서 조절이 이루어지는 것을 알 수 있었다. Glycinin is the most abundant storage protein in soybean. A molecule of glycinin is composed of 6 subunits each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one. These polypeptides are synthesized as a precursor which is cleaved to yield A- and B-polypeptides. To study the structure and expression mechanism of the gene, isolation of cDNA clones for soybean glycinin was attempted by immunological screening of a soybean cDNA library with antibodies. The cDNA expression library was constructed with double stranded cDNA of polyadenylated mRNA into ${\lambda}$gt11 expression vector. Antibodies to A- and B-polypeptides of glycinin subunit which correspond to N- and C-terminal domain of mRNA translate, respectively, were employed. Eight clones for glycinin, 4 with anti-A and 4 with anti-B polypeptide antibody, were isolated by immunoscreening. The clone ${\lambda}$G1A1 with 1186 bp insert was characterized to be a partial cDNA clone for glycinin subunit $A_2B_{1a}$ by nucleotide sequencing analysis and the expression in E. coli. It encodes a half of $A_2$- and entire $B_{1a}$-polypeptide. The mRNA size of glycinin was demonstrated to be 1.8 kb by Northern blot analysis. It was expressed only in seeds maintaining tissue-specific expression pattern, which was controlled at the level of transcription rather than at translation.

대두 Bowman - Birk trypsin isoinhibitor PI Ⅳ의 cDNA 유전자 분리

안중훈,김희진,최양도,김수일 ( Joohg Hoon Ahn,Hee Jin Kim,Yang Do Choi,Su Il Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Three different kinds of proteinase inhibitors are known in soybeans; Kunitz trypsin inhibitor, Bowman-Birk trypsin inhibitor (BBTI) and its isoinhibitors. A cDNA clone for the soybean trypsin inhibitor was isolated from a soybean λgt11 cDNA library by plaque hybridization with a oligonucleotide probe which corresponds to the N-terminal region of the BBTI. The cDNA clone λB13 was 446bp long and the nucleotide sequencing reveals that there were one open reading frame of 243 bp, the 5` leader sequences of 70 bp and the 3`-untranslating region of 133 bp in the cDNA clone λB13. Nucleotide sequences at the 5` and 3` noncoding regions were highly homologous, respectively, to those of the BBTI and isoinhibitor C-2. Compared the deduced amino acid sequences to the amino acid sequences of proteinase inhibitor, it corresponds to the Bowman-Birk trypsin isoinhibitor PI IV. Sequences around the reactive site of Arg-Ser were repeated twice, giving the double headed structure. Northern blot analysis demonstrated that the size of the mRNA was about 700 nucleotides. It was expressed only in soybean seed maintaining tissue specific gene expression pattern. Expression level was increased with the maturation of seed and reached maximum in full size seed. It suggests that the expression of the isoinhibitor PI IV could be regulated at the transcriptional level.

Molecular Cloning and Nucleotide Sequencing of cDNA encoding Soybean Bowman-Birk Trypsin Isoinhibitor PI IV

안중훈,김희진,최양도,김수일,Ahn, Joong-Hoon,Kim, Hee-Jin,Choi, Yang-Do,Kim, Su-Il 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

대두의 cDNA 유전자 은행을 선별한 결과 Bowman-Birk trypsin isoinhibitor PI IV의 cDNA 유전자를 분리하였다. Probe 는 Bowman-Birk trypsin inhibitor (BBTI)의 아미노 말단 부위에 해당하는 oligonucleotide를 합성 사용하였다. 이 유전자의 크기는 446 bp 였다. 염기서열을 결정한 결과 243 bp 의 open reading frame 과 70 bp 의 5'-leader sequence 그리고 133 bp의 3'-untranslating sequence로 이루어져 있었다. 5'-과 3'-noncoding 부위 각각의 염기서열은 BBTI 와 isoinhibitor C-2의 경우와 매우 유사하였다. 이 유전자에 의해 code되는 단백질은 6 개 아미노산의 signal peptide 를 포함한 Bowman-Birk iscinhibitor PI IV로 나타났다. 아미노산 Arg-Ser을 포함한 활성부 위는 두번 반복하고 있어서 double headed structure를 이루고 있었다. Northern blot을 실시한 결과 이 유전자의 mRNA는 길이가 약 700 nucleotide 였으며 대두 종자에서만 발현되었다. 종자가 성숙하면서 유전자 발현은 증가하여 완숙된 대두에서 최고치를 나타내었다. 이 결과 PI IV는 전사 단계에서 발현 조절이 이루어지는 것을 알 수 있다. Three different kinds of proteinase inhibitors are known in soybeans; Kunitz trypsin inhibitor, Bowman-Birk trypsin inhibitor (BBTI) and its isoinhibitors. A cDNA clone for the soybean trypsin inhibitor was isolated from a soybean ${\lambda}$gt11 cDNA library by plaque hybridization with a oligonucleotide probe which corresponds to the N-terminal region of the BBTI. The cDNA clone ${\lambda}$ B13 was 446bp long and the nucleotide sequencing reveals that there were one open reading frame of 243 bp, the 5' leader sequences of 70 bp and the 3'-untranslating region of 133 bp in the cDNA clone ${\lambda}$B13. Nucleotide sequences at the 5' and 3' noncoding regions were highly homologous, respectively, to those of the BBTI and isoinhibitor C-2. Compared the deduced amino acid sequences to the amino acid sequences of proteinase inhibitor, it corresponds to the Bowman-Birk trypsin isoinhibitor PI IV. Sequences around the reactive site of Arg-Ser were repeated twice, giving the double headed structure. Northern blot analysis demonstrated that the size of the mRNA was about 700 nucleotides. It was expressed only in soybean seed maintaining tissue specific gene expression pattern. Expression level was increased with the maturation of seed and reached maximum in full size seed. It suggests that the expression of the isoinhibitor PI IV could be regulated at the transcriptional level.