Nonenzymatic Cleavage of Escherichia coli Glutamine Synthetase by Dithiothreitol and Iron(III)

전덕영,김강화,변시명,Jhon, Deok-Young,Kim, Kang-Hwa,Byun, Si-Myung 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4

대장균 글루타민 합성효소에 대한 디티오트레이톨과 철 (III)이온에 의한 비효적 절단반응의 최적조건은 이 효소의 농도가 단량체로서 0.06 mM 일 때 디티오트레이톨 5 mM, 철 (III) 0.1 mM, pH 7.0, $37^{\circ}C$, 반응시간은 4시간이었다. 절단양상은 기질 및 기질유사체인 글루타민, 글루타민산, L-메치오닌 술폭시민 등 리간드의 첨가에 의하여 크게 변화되었다. 그러나 이들 화합물은 동일한 절단조건에 의해서 절단되는 피루브산 키나제에 대해서는 그 고유한 절단양상을 변화시키지 않았다. 이들 결과는 대장균 글루타민 합성효소의 디티오트레이톨과 철 (III) 이온에 의한 절단이 효소의 활성부위에서 일어남을 시사한다. Optimum reaction condition for the cleavage of Escherichia coli glutamine synthetase in the presence of oxygen, iron(III), and dithiothreitol has been established. The condition is 5 mM dithiothreitol, 0.1 mM iron(III), pH 7.0,$37^{\circ}C$, and 4hours of reaction time, when the enzyme concentration is 0.06 mM as subunit basis. The cleavage pattern strongly depends on the addition of ligands of the enzyme, glutamic acid, glutamine, L-methionine-S-sulfoximine, to the reaction mixture while the ligands do not change the cleavage pattern of rabbit muscle pyruvate kinase which is also attacked by the cleavage system. The results indicate that the cleavage reaction occurrs at the polypeptide segments which consist the active site of the glutamine synthetase.

The Presence of an Antioxidant Protein to Inhibit Enzyme Inactivation by an Ascorbate/Fe(III)/$O_2$, Mixed-function Oxidation System in Yeasts

김강화,조남철,전덕영,Kim, Kang-Hwa,Cho, Nam-Chul,Jhon, Deok-Young Korean Society for Biochemistry and Molecular Biol 1990 한국생화학회지 Vol.23 No.3

Saccharomyces와 Candida의 두 종류 효모 추출물 중에서 아스코르브산/철(III)/산소 혼합기능 산화계를 보다 선택적으로 억제하는 항산화 활성의 존재를 확인하였다. 두 효모의 조단백질을 DEAE관을 이용한 고속액체 크로마토그래피 방법으로 분리하였을 때 디티오트레이톨이나 아스코르브산을 전자공여체로 하는 혼합기능 산화계에 의한 효소의 불활성화를 억제하는 각각 세 종류의 항산화 활성도가 검출되었다. 이 중 두 효모 모두에서 동일하게 0.22M 염화칼륨 농도에서 용출되는 분획 중에 포함되어 있는 항산화 활성도는 아스코르브산/철(III) 산소 혼합기능 산화계를 선택적으로 억제하였다. 이는 디티오트레이톨/철(III)/산소와 아스코르브산/철(III)/산소 두 혼합기능 산화계를 같은 정도로 억제하는 카탈라제나 S. cerevisiae에서 정제 보고된 티올기 특이적 항산화 단백질로서 디티오트레이톨/철(III)/산소 혼합기능 산화계만을 억제하는 활성도와는 전혀 다른 것으로서 카탈라제 활성도를 나타내지 않았고 티올기 특이적 항산화 단백질에 대한 항체와 반응하는 단백질도 함유하지 않았다. 따라서 이들 효모 중에는 카탈라제와 티올기 특이적 항산화 단백질외에 아직 보고되지 않은 새로운 종류의 항산화 단백질이 있는 것으로 추정된다. Antioxidant activity which specifically inhibits the mixed-function oxidation(MFO) system comprised of ascorbate, Fe(III), and $O_2$ was found from both extracts of Saccharomyces cerevisiae and Candida pseudotropicalis. From crude extracts of the yeast three different antioxidant activities against MFO systems containing dithiothreitol (DTT) or ascorbate as an electron donor could be separated by a HPLC-DEAE column chromatography. Antioxidant activity of the fraction eluted at 0.22 M potassium chloride from both yeast preparations selectively inhibited the ascorbate/Fe(III)/$O_2$ MFO system. It differed from catalase activity which inhibits DTT/Fe(III)/$O_2$ and ascorbate/Fe(III)/$O_2$ MFO systems with similar extent and thiol-specific antioxidant activity which was reported from S. cerevisiae and inhibits DTT/Fe(III)/$O_2$ MFO system specifically. The active fraction showed no catalase activity and did not carry immunoreactive protein which specifically reacts with antibody against thiol-specific antioxidant protein. The results suggest that there is another type of antioxidant protein besides catalase and thiol-specific antioxidant protein in yeast.

Purification and Characterization of Polyol Dehydrogenase from Gluconobacter melanogenus

조남철,김강화,전덕영,Cho, Nam-Chul,Kim, Kang-Hwa,Jhon, Deok-Young 생화학분자생물학회 1990 한국생화학회지 Vol.23 No.2

Gluconobacter melanogenus의 세포질과 세포막으로부터 NAD(P) 비요구성 폴리올 탈수소 효소를 분리하여 순차적으로 CM-셀롤로오즈의 통과, 풀리에칠렌 글리콜 침전, DEAE-Sephacel 컬럼의 통과, SP-5 PW 컬럼을 이용한 HPLC를 행하여 정제하였다. 정제된 효소는 소단위의 구조, 흡수 스펙트럼이 서로 같았고 기질특이성은 유사하였다. 이 효소는 시토크륨 C특유의 552, 522, 418 nm에서 흡수 피크를 갖는 흡수 스펙트럼을 보였다. 효소 중의 산화된 시토크롬 성분은 기질인 만니톨에 의하여 빠르게 환원되었다. 두 효소는 모두가 SDS-전기영동에 의하여 분자량이 63 K, 52 K, 20.5 K인 소단위로 구성되어 있음을 보였다. 그 중에서 분자량이 52 K인 소단위가 산화된 시토크롬 C와 같은 흡수 스펙트럼을 나타냈다. 이 효소는 제한된 기질특이성을 보였는데 D-lyxo 구조를 갖는 기질인 만니톨이나 솔비톨에 대하여만 효소활성을 보였으며 특히 만니톨에 대한 활성도가 컸다. From cytoplasm and cell membrane of Gluconobacter melanogenus NAD(P) independent polyol dehydrogenases were purified by polyethylene glycol precipitation, conventional CM-cellulose and DEAE-Sephacel column, and HPLC SP column chromatography. The two enzymes gave same subunit structure, same absorption spectrum, and similar substrate specificity. The enzyme showed a characteristic absorption spectrum of cytochrome c showing maxima at 552, 522, and 418 nm. The oxidized cytochrome component of the enzyme was rapidly reduced by a substrate, D-mannitol. Based on SDS-PAGE, the purified enzyme was composed of three different subunits having Mr of 63 K, 52 K and 20.5 K, respectively. The second subunit (Mr 52 K) showed oxidized cytochrome c absorption spectrum. The enzyme had a restricted substrate specificity toward the polyols having D-lyxo configuration like D-mannitol and D-sorbitol showing higher activity on D-mannitol.

효모 중 아스코르브산을 포함하는 혼합기능 산화계를 억제하는 새로운 항산화 단백질의 존재에 관한 연구

김강화,조남철,전덕영 ( Kang Hwa Kim,Nam Chul Cho,Deok Young chon ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.3

Antioxidant activity which specifically inhibits the mixed-function oxidation(MFO) system comprised of ascorbate, Fe(III), and O₂ was found from both extracts of Saccharomyces cerevisiae and Candida pseudotropicalis. From crude extracts of the yeast three different antioxidant activities against MFO systems containing dithiothreitol (DTT) or ascorbate as an electron donor could be separated by a HPLC-DEAE column chromatography. Antioxidant activity of the fraction eluted at 0.22 M potassium chloride from both yeast preparations selectively inhibited the ascorbate/Fe(III)/O₂ MFO system. It differed from catalase activity which inhibits DTT/Fe(III)/O₂ and ascorbate/Fe(III)/O₂ MFO systems with similar extent and thiol-specific antioxidant activity which was reported from S. cerevisiae and inhibits DTT/Fe(III)/O₂ MFO system specifically. The active fraction showed no catalase activity and did not carry immunoreactive protein which specifically reacts with antibody against thiol-specific antioxidant protein. The results suggest that there is another type of antioxidant protein besides catalase and thiol-specific antioxidant protein in yeast.

Gluconobacter melanogenus 폴리올 탈수소 효소의 정제 및 특성에 대한 연구

조남철,김강화,전덕영 ( Nam Chul Cho,Kang Hwa Kim,Deok Young Jhon ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.2

From cytoplasm and cell membrane of Gluconobacter melanogenus NAD(P) independent polyol dehydrogenases were purified by polyethylene glycol precipitation, conventional CM-cellulose and DEAE-Sephacel column, and HPLC SP column chromatography. The two enzymes gave same subunit structure, same absorption spectrum, and similar substrate specificity. The enzyme showed a characteristic absorption spectrum of cytochrome c showing maxima at 552, 522, and 418 nm. The oxidized cytochrome component of the enzyme was rapidly reduced by a substrate, D-mannitol. Based on SDS-PAGE, the purified enzyme was composed of three different subunits having Mr of 63 K, 52 K and 20.5 K, respectively. The second subunit (Mr 52 K) showed oxidized cytochrome c absorption spectrum. The enzyme had a restricted substrate specificity toward the polyols having D-lyxo configuration like D-mannitol and D-sorbitol showing higher activity on D-mannitol.

Gluconobacter melanogenus 폴리올 탈수소 효소의 정제 및 특성에 대한 연구

김강화,조남철,전덕영 생화학분자생물학회 1994 BMB Reports Vol.17 No.3

From cytoplasm and cell membrane of Gluconobacter melanogenus NAD(P) independent polyol dehydrogenases were purified by polyethylene glycol precipitation, conventional CM-cellulose and DEAE-Sephacel column, and HPLC SP column chromatography. The two enzymes gave same subunit structure, same absorption spectrum, and similar substrate specificity. The enzyme showed a characteristic absorption spectrum of cytochrome c showing maxima at 552, 522, and 418 nm. The oxidized cytochrome component of the enzyme was rapidly reduced by a substrate, D-mannitol. Based on SDS-PAGE, the purified enzyme was composed of three different subunits having Mr of 63 K, 52 K and 20.5 K, respectively. The second subunit (Mr 52 K) showed oxidized cytochrome c absorption spectrum. The enzyme had a restricted substrate specificity toward the polyols having D-lyxo configuration like D-mannitol and D-sorbitol showing higher activity on D-mannitol.