화학적 변형에 의한 Trichoderma Viride Cellulase의 β-Glucosidase특성연구

고은희,이혜숙 德成女子大學校 1992 德成女大論文集 Vol.21 No.-

In the light of the wide industrial significance of cellulase, in is requested to obtain the basic (research) data on the chemical and physical characteristics of the each component of the enzyme. Chemical modification of the amino acid residues of T. viride cellulase was carried out as a means of illustrating the enzymatich mechanisem of β-Glucosidase. 5.5'-dithiobis-2-nitrobenzoate (DTNB) and n-acetylimidazole(NAI) were chosen as the modifying agents for the free -SH group of cystein residues and the tyrosine residues of the enzyme respectively. DTNB altered neither the enzyme activity nor the absorbance at 412nm, suggesting that cysteins of the enzyme are not likely to exist as the free -SH form. The reactions with NAI resulted in the reversible chemical modification of the enzyme and decrease of the enzyme activity. The results imply that the tyrosine might be involved in the active sites of the β-Glucosidase.

쥐 뇌 Arylsulfatase Bm의 분리 및 특성연구

고은희 德成女子大學校 1985 德成女大論文集 Vol.14 No.-

쥐 뇌로부터 Arysulfatase Bm을 부분정제하여 그 특성을 연구하였다. 정제는 황산암모늄 분별침전, DEAE-Cellulose 크로마토그라피, Sephadex G-200겔 여과법, ConA-Sepharose 4B 친화크로마토그라피 단계를 거쳐 이루어졌으며 약 100배의 정제도와 17%의 회수율을 나타냈다. Arylsulfatase Bm의 일반적 특성을 p-nitrophenyl sulfate를 기질로 사용하여 연구되었다. 기질의 농도가 각각 2.5mM, 5mM, 25mM일때 효소활동도와 반응시간의 관계는 75분까지는 직선비례로 나타났다. 반응속도와 기질농도의 관계는 비정상적인 두개의 상을 갖는 곡선으로 나타났으며 기질의 농도가 대략 40mMdlfEo 반응속도는 감소를 보였다가 다시 증가하였다. 여기에서 나타나는 비정상적인 속도대 농도곡선은 현 시점에서는 설명할 수가 없으며 40mM 농도까지인 첫번째 곡선 부분에서 얻어진 속도 매개 변수 V??과 K??은 각각 22.73μ/min/mg 단백질, 83.5mM이었다. Arylsulfatase Bm은 pH 5.6과 6.4 두개의 최적 pH를 갖는 것으로 나타났다. Ca², Mn², Mg²양이온은 효소작용을 억제했으며 생성물중의 하나인 황산음이온도 효소작용을 저해 하는 것으로 나타났다. 황산이온의 영향은 일반적인 생성물에 의한 효소활동도 저해라고 생각할 수 있겠으나 또 하나의 생성물이라고 할 수 있는 페놀은 아무런 영향을 나타내지 않았다. 계면활성제 SDS와 Triton X-100는 효소작용을 활성화 시켰으며 특히 Triton X-100는 그 농고가 0.5%일 때 10배 이상의 활동도 증가를 보여주어 특기할만하다. 이들 실험결과들은 앞으로의 Arylsulfatase Bm의 생화학적 성질을 자세히 규명하기 위한 연구의 기초자료로 사용될 수 있을 것으로 사려된다. Arylsulfatase(Arylsulfate sulfohydrolase, EC. 3.1.6.1.) Bm from rat brain was partially purified and characterized. The purification steps involved ammonium sulfate fractionation, DEAE-cellulose ion exchange chromatography, gelfiltration on Sephades G-200, and concanavalin A-Sepharose 4B affinity chromatography. Arylsulfatase Bm was purified 100-fold with 17% recovery. The general properties of Arylsulfatase Bm was investigated with p-nitrophenyl sulfate as substrate. When the concentration of substrate was either 2.5mM, 5mM, or 25mM, the activity of the enzyme vs. the incubation time showed a linearity up to the 75min. The reaction rate vs. the suvstrate concentration exhibits an unusual shaped curve with two distinctive phases. Invariably the reaction rate of enzyme leveled off at about 40mM substrate and again increased steadily up to 100mM. The reason for this unusual rate vs. concentration relationship cannot be explained satisfactorily at this point. The kinetic parameters M?? and K?? were found to be 22.73μmole/min/mg protein and 83.5mM respectively for the first phase of the rate vs. concentration curve. Arylsulfatase Bm has two optimum pH, 5.6 and 6.4 under the experimental condition. Cations such as Ca²??, Mn²??, and Mg²?? inhibit the enzyme activity. Sulfate anion, which is one of the products, inhibits the enzyme activity probably due to the product inhibition. But phenol has no effect on the enzyme activity under the experimental conditions employed. Detergents, SDS and Triton X-100, exhibit a remarkable activation of the enzyme. A cioser investigation of the effect of Triton X-100 on the enzyme activity showed that the enzyme was activated approximately 10 fold when the incubation mixture contained 0.5% Triton X-100. The data presented here can be utilized as basic materials for the biochemical studies of Arylsulfatase Bm in order to elucidate the biochemical characteristics of this enzyme.

ABSTRACTS ; Posters : Studies on Biochemical Characteristics of yeast ALS

Eun Hie Koh,Sun Young Kim 생화학분자생물학회(구 한국생화학분자생물학회) 1995 추계학술대회집 Vol.- No.-

Session E : Enzymes : Mechanism of Action and Regulation : Purification and Characterization of Acetolactate Synthase from Baker ' s Yeast

Eun Hie Koh,Soo Mee Song 생화학분자생물학회(구 한국생화학분자생물학회) 1993 추계학술대회집 Vol.- No.-

Catalytic Properties of Phospholipase D using Phosphatidic Acid as an Activator

Eun-hie Koh,Myung-Un Chol,Kwanyoung Jung Korean Chemical Society 1989 Bulletin of the Korean Chemical Society Vol.10 No.6

The effects of phosphatidic acid(PA) on the activity of phospholipase D were examined in detail. The enzyme activity was examined in the liposome system containing phosphatidylcholine and PA, which was suspended in a desired buffer solution by ultrasonication. The substrate of large unilamella vesicle (LUV) state by ultrasonication was more effective on the enzyme activity than that of multilamella vesicle(MLV) by water-bath type sonication. The most effective molar ratio of PC-PA liposome for enzyme activity was found to be 1:0.7. The other optimum conditions were found 5 mM $Ca^{2+}$ ion, pH 6.6, and incubation temperature of $27^{\circ}C. K_m \;and \;V_{max}$ values were estimated to be 1.43 mM and 0.8 $nmole/min/{\mu}g$ protein respectively. These properties in a PC-PA liposome system were compared with those in a PC-SDS mixed micelle system. The effects of other phospholipids and organic phosphates on the enzyme activity were also examined.

신경전달물질 대사에 관여하는 효소들

고은희(Eun Hie Koh),정태숙(Tae Sook Jeong),최명언(Myung Un Choi) 생화학분자생물학회(구 한국생화학분자생물학회) 1988 생화학총설집 Vol.2 No.-

씨클 적혈구의 씨클링과 헤모글로빈 S의 겔화에 대한 이산화탄소의 영향

고은희 ( Eun Hie Koh ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.4

Effects of carbon dioxide on the gelation of Hb S and the sickling of sickle erythrocytes were studied. Increasing the Pco, over the range of 0∼76 mmHg under total deoxygenation increases the sickling of sickle erythrocytes within the pH range of 6.8∼8.0. The enhancement of sickling was gradual as a function of Pco₂ at pH 8.0, while essentially complete sickling was achieved at Pco₂ as low as 10 mmHg at pH 6.8. In the absence of 2, 3-diphosphoglycerate (DPG), decreases of 2 gram percent in minimum gel concentration (MGC) were observed when 45 mmHg CO₂ was added to deoxy Hb S solutions at pH 7.2∼7.6, showing that the effect of CO₂ on the sickling of sickle cells is a direct manifestation of the enhanced gelation of carbamated Hb S. The observed effect of CO₂ favoring gelation may be explained in terms of the conformational changes resulting from the formation of the Hb S-carbamate adduct.

Tricoderma viride cellulase의 부분정제와β-Glucosidase 활동도에 대한 생화학적 특성 연구

고은희,김효진 덕성여자대학교 자연과학연구소 1996 자연과학 논문집 Vol.2 No.-

The biochemical characteristics of commercially available T.viride cellulase were studied and the optimum temperature was determined to be 50℃, 55℃, and 60℃ for Avicelase, CMCase, and β-Glucosidase respectivity. The optimum pH of all three enzyme activity was 5.0. The fraction showing β-Glucosidase activity was purified by 1.6 fold from cation-exchange chromatography on SP-Sephadex C-50. Three protein bands were obtained on SDS-polyacrylamide gel-electrophoresis corresponding to MW. 76.000, 65.000, and 60.000 respectively. The enzyme was treated with NAI, a known chemical modifying agent of the tyrosine side chain, to study the role of the tyrosine residues in the catalytic activity. The enzyme activity decreased by 40% compared to the unmodified form. Moreover, the degree of modification increased in the presence of urea. These results suggest that tyrosine residue(s) take part in the catalytic function of the β-Glucosidase activity.

Aspergillus niger cellulase의 부분 정제 및 β-glucosidase의 생화학적 특성 연구

고은희,김영숙 덕성여자대학교 자연과학연구소 1995 자연과학 논문집 Vol.1 No.-

β-Glucosidase was purified 50-fold from the commercially obtained Aspergillus niger cellulase to give a single band on SDS-polyacrylamide electrophoresis(M.W. 91,700). Amino acid analysis of the purified portion showed high Alanine and Lysine content. A chemical modification of the β-glucosidase with Lysine-specific chemical modifying agent, 2,4-dinitrofluorobenzene showed a gradual activity loss on incubation up to 24 hours. The enzyme lost its activity almost completely after 24 hours. The results suggest that Lysine residue(s) are involved in a crucial role, either directly or indirectly in the β-glucosidase enzyme activity.

HepG2 세포의 포스포리파제 D 활성과 자유 지방산 방출에 대한 디프테리아 독소의 영향

고은희,Koh, Eun-Hie 대한화학회 2015 대한화학회지 Vol.59 No.1

본 연구에서는 디프테리아 독소가 세포막의 지질에 미치는 영향을 알아보기 위해 HepG2 세포에서 포스포리파제 D와 유리된 지방산(Free fatty acid)의 변화를 살펴보았다. 지질변화는 pH 5.1에서 최고 값을 나타냈으며, 이 pH에서 포스포리파아제 D의 활성을 3.5배 가량, 유리된 지방산의 방출은 5배 정도 증가되었다. 이는 디프테리아 독소가 세포 안으로 들어가는 과정에서 세포막이 교란되어 재배열되었음을 시사한다. 한편 세포막을 무작위로 교란시키는 디지토닌의 영향이 디프테리아 독소의 그것보다 중성 pH에서 4배 이상 상당히 높게 나타난 것으로 미루어 보아 디프테리아 독소의 영향이 상대적으로 선택적인 교란 현상인 것으로 보여진다. 이런 세포막 교란의 연유를 밝히고자 세포막 구멍 형성 저해제인 cibacron blue와 세포막 융합 펩티드를 갖고 있는 hemagglutinin의 영향을 검토하였다. Cibacron blue는 디프테리아 독소에 의한 지질 변화를 50% 정도 저해시켰으며, hemagglutinin에 의한 지질변화는 디프테리아 독소의 그것과 유사함을 관찰 할 수 있었다. 이들 결과들은 디프테리아 독소에 의한 세포막 교란이 구멍형성과 독소의 소수성 펩티드가 세포막에 삽입되는 과정이 서로 연계되어 있음을 암시한다. 그 외 일련의 실험으로 디프테리아 독소가 세포막을 통과하는 과정에서 HepG2 세포의 투과성은 상승시켰으나, 세포의 생존능력은 상당히 높게 유지되었고 DNA 토막내기 같은 세포의 괴사는 일어나지 않았다. 이런 조건하에서 디프테리아 독소는 산성 pH에서 HepG2 세포의 지질의 변화를 가져 온다는 것을 밝힐 수 있었다. The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.