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.

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.

Life Style SPA 기업의 성공전략

김강화 한국생활과학회 2008 한국생활과학회 학술대회 Vol.2008 No.동계

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.

Kinetic Properties of the Dye-Coupled Cytoplasmic Polyol Dehydrogenase from Gluconobacter melanogenus

김강화,이현재,Kang-Wha Kim,Hyun-Jae Lee Korean Chemical Society 1980 대한화학회지 Vol.24 No.4

G. melanogenus로부터 분리한 폴리올 탈수소 효소는 이미 알려진 바의 다른 폴리올 탈수소 효소와는 달리 조효소로서 2,6-dichlorophenolindophenol (DPIP)와 같은 인위적 전자 수용체를 필수로 요구하고 있음으로 이 특수 효소의 반응메카니즘을 반응속도론적 연구를 통하여 규명코저 시도하였으며, 폴리올 산화반응에 대한 초기속도 측정실험과 효소반응 산물인 케토산에 의한 저해 실험을 통하여 이 반응은 Ping-Pong Bi-Bi형의 반응메카니즘으로 진행됨을 확인하였다. 따라서 두 기질 즉 포리올로서 D-mannitol 및 전자수용체로 DPIP가 효소에 의하여 반응이 진행될 경우 D-mannitol이 우선 효소와 작용하며 첫 반응산물로서 해당하는 케토산인 D-fructose가 생성될 것으로 기대되며 이 반응이 전체 반응속도를 조절하는 과정일 것이라고 추측하였다. A steady-state kinetic study on a dye-coupled cytoplasmic polyol dehydrogenase from G. melanogenus was carried by the initial velocity measurements in the direction of the polyol oxidation and the product inhibition by D-fructose. For the initial rate experiments, D-mannitol and D-sorbitol were employed as the specific polyol substrates and 2,6-dichlorophenolin-dophenol (DPIP) as the specific cofactor substrate for the enzyme. When the polyol and DPIP were examined by varying one of substrates and by fixing the second, the corresponding reciprocal plots showed the typical parallel pattern. This suggests that the enzyme from G. melanogenus proceeds by a Ping Pong Bi-Bi mechanism in which the polyol may account as the first reactant-in, and the ketose formed as the first product-out, respectively. The product inhibition patterns obtained by D-fructose (one no-inhibition, one non-competitive, and two competitive) may also provide an additional conformatory evidence for the above mechanism. Based on the kinetic parameters obtained, it was also suggested that the rate-limiting step in the direction of polyol oxidation is associated with the release of the ketose from the Enzyme${\cdot}$Polyol complex.

Gluconobacter melanogenus 로 부터 새로운 포리올 탈수소 효소의 분리 정제와 효소 특성에 관한 연구

김강화,임옥재,이현재 ( Kang Wha Kim,Ok Jae Im,Hyun Jae Lee ) 생화학분자생물학회 1980 BMB Reports Vol.13 No.3

A new form of the cytoplasmic polyol dehydrogenase having a distinct cofactor and substrate specificity was isolated from the cell free extracts of the sorbitol grown cells of G. melanogenus, and purified partially by CM-cellulose and Sephadex G-100 columns. The enzyme was found to be acted as a typical polyol dehydrogenase catalyzing the oxidation of the polyols to the ketoses, but unlike the other known cytoplasmic dehydrogenases, its catalytic activity was demonstrated only in the presence of an artificial electron acceptor like DPIP. The enzyme did not showed any requirement of pyridine nucleotide coenzymes, nor the molecular oxygen for the polyol oxidation. In the presence of DPIP, however, the enzyme was active and showed a very limitted substrate specificity toward the polyols having D-lyxo configuration such as D-mannitol and D-sorbitol. The K_m values for the tested polyol substrates were found to be about 10^(-1)M, and the optimum enzyme activity was obtained at pH 5. 5. Stoichiometric composition of the polyol and DPIP for this enzyme catalyzed reaction showed a linear quantitative relationship between the rates of the ketose formation and the DPIP reduction. The characteristic mode of the enzyme action was also studied by some kinetic and inhibition experiments with several substrate analogues.

Purification and Characterization of the New Dye Coupled Cytoplasmic Polyol Dehydrogenase from Gluconobacter Melanogenus

김강화,임옥재,이현재,Kim, Kang-Wha,Im, Ok-Jae,Lee, Hyun-Jae 생화학분자생물학회 1980 한국생화학회지 Vol.13 No.3

초산박테리아 속의 하나인 G. melanogenus로부터 새로운 포리올 탈수소효소를 얻어 정제하였으며, 이 효소의 특성을 연구 검토해 본 결과 이 효소는 지금까지 알려진 바의 포리올 탈수소효소와는 달리 조효소로서 $NAD^+$나 $NADP^+$ 등을 필요로 하지 않는 대신 2, 6-dichlorophenolindophenol(DPIP)와 같은 인위적인 전자 수용체와 직접 연결되어 있음을 알았다. 한편 효소의 기질 특이성에 있어서도 D-Mannitol과 D-Sorbitol 같은 D-lyxo 형의 구조를 갖는 포리올에 대하여만 특이하게 작용하여 산화적 촉매 활성도를 보임으로서 해당하는 케토산인 D-Fructose와 L-sorbose를 생성시킴을 알았다. 그밖에도 본연구에서는 이 새로운 효소의 일반적 특성 즉, 효소의 안정도, 최적활성도 및 기질에 대한 친화력 등에 대하여도 검토하였으며. 이 효소의 생리적 본질에 대하여 서로 고찰해 보았다. A new form of the cytoplasmic polyol dehydrogenase having a distinct cofactor and substrate specificity was isolated from the cell free extracts of the sorbitol grown cells of G. melanogenus, and purified partially by CM-cellulose and Sephadex G-100 columns. The enzyme was found to be acted as a typical polyol dehydrogenase catalyzing the oxidation of the polyols to the ketoses, but unlike the other known cytoplasmic dehydrogenases, its catalytic activity was demonstrated only in the presence of an artificial electron acceptor like DPIP. The enzyme did not showed any requirement of pyridine nucleotide coenzymes, nor the molecular oxygen for the polyol oxidation. In the presence of DPIP, however, the enzyme was active and showed a very limitted substrate specificity toward the polyols having D-lyxo configuration such as D-mannitol and D-sorbitol. The $K_m$ values for the tested polyol substrates were found to be about $10^{-1}M$, and the optimum enzyme activity was obtained at pH 5.5. Stoichiometric composition of the polyol and DPIP for this enzyme catalyzed reaction showed a linear quantitative relationship between the rates of the ketose formation and the DPIP reduction. The characteristic mode of the enzyme action was also studied by some kinetic and inhibition experiments with several substrate analogues.

대장균 페리틴의 항산화 활성

이송미,김강화 全南大學校家政科學硏究所 1999 生活科學硏究 Vol.9 No.-

Ferritin (Ftn), an iron storage protein, takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The antiocxidant activity of Ftn against the metal catalyzed oxidation systems comprised of ferric ion or cupric ion and reducing agents was investigate. Ftn of Escherichia coli was overexpressed and purified by ion-exchange and gel chromatographies. The protein inhibited the inactivation of glutamine synthetase caused by metal catalyzed oxidation system comprised of either dithiothreitol or ascorbate as a reducing agent. However, removal activity of H₂O₂by Ftn wasn't detected.

육미지황탕이 흰쥐의 뇌손상 회복에 미치는 영향

김방울,김경선,전홍열,강화정,김정상,홍석,김용진,Kim, Bang-Oul,Kim, Kyoung-Sun,Jeon, Hong-Yeol,Kang, Hwa-Jeong,Kim, Jeong-Sang,Hong, Seok,Kim, Yong-Jin 대한한방내과학회 2002 大韓韓方內科學會誌 Vol.23 No.2

Objectives : This study was designed to investigate the effects of Yukmijihwang-tang on contusion of the mice induced with medicine. Methods : I observed the effects of light and electron microscopes. and examined hematological changes and VEGF-immunohistochemistry. Results : Hematology: Leukocytes were increased significantly in a control group of mice compared with the experimental group. Light microscope : A few neurons were condensed in the 7-day experimental group, but condensed remarkedly in the 3-day control group. Most glial cells were observed in the 3-day experimental group. Edema and dilatation of vessels occurred significantly in the 3-day control group, and these results occurred weakly in the 7-day experimental group. VEGF-immunohistochemistry : VEGF-immunohistochemical reactivity for the glial cells was the highest in the 3-day experimental group, and immunoreactivity for the vessels and neurons highly increased in the 7-day experimental group. Electron microscope : In the 3-day control group, protoplasmic astrocytes concerned with angiogenesis contained weakly developed rough endoplasmic reticulum. and a few of glial filaments were observed. In the 7-day experimental group, the bundles of glial filaments were found in the cytoplasmic process of astrocytes. Conclusion : medication using Yukmijihwang-tang of mice contused by medical stress is highly effective in inflamatory response, curing cell damage and angiogenesis.

중추신경계에서 타올-특이 항산화단백의 분포

김요식,김병채,조기현,김세종,박사훈,이기형 김강화,배춘상 대한신경과학회 1995 대한신경과학회지 Vol.13 No.1