The Peroxidase Activity of Rat Brain : Subcellular and Regional Distributions

최명언,Choi, Myung-Un 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.4

쥐 뇌의 Peroxidase 활성도를 o-dianisidine을 이용 분광학적 방법으로 측정했다. 유사 peroxidase를 제거하기 위해 기본적인 효소측정의 여건을 살폈으며 이에 따라 세포내 및 뇌의 여섯 부위에서 peroxidase 활성도를 검토했다. 대부분의 활성도는 particulated fraction에서 발견됐으며 microsome의 고유 활성도는 mitochondria에 비해 약 두배였다. Microsome 활성도의 뇌 부위별 분포는 상당히 변화가 있어 medulla oblongata에서 가장 높았고 striatum에서는 가장 낮은 활성도를 보여 주었다. 뇌 peroxidase의 가능한 역활에 대하여 간단히 평가했다. The peroxidase activity of rat brain was determined by spectrophotometrically using o-dianisidine as substrate. Basic assay conditions were optimized in order to eliminate the pseudoperoxidase activity. The peroxidase activities were then examined in subcellular fractions as well as in six different regions of the brain. It was found that a major part of the activity was associated with particulated fractions. The specific activity of the microsomal enzyme was roughly twice as high as that of the mitochondrial. The regional distribution of the microsomal activity was quite variable. The highest activity was found in the medulla oblongata and lowest in the striatum. The possible role of the brain peroxidase was birefly assessed.

Kinetic Behavior of Solubilized Microsomal Cholesterol Ester Hydrolase of Rat Brain

최명언,Choi, Myung-Un 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.4

쥐 뇌 microsome의 cholesterol ester hydrlase를 좋은 안정성과 수득율로 용해 시켰다. 최적 용해는 0.75%의 Triton X-100을 포함하는 pH7.6의 TrisHCl 완충액 1 ml당 10 mg의 microsome을 섞어 균질화하고 고속원심분리하여 이루어졌다. 이렇게 용해된 효소는 mlcrosom에 부착되었을 때 보다 더 외부에서 넣어준 지방질에 의해 그 활성도가 좌우됐으며 반응에 필요한 Triton X-100 농도는 크게 줄어 들었다. Triton X-100, 기질 및 효소의 농도를 독립적으로 변화시키면서 한 일련의 실험으로, 이 효소에 대한 계면활성제의 활성화 효과는 계면활성제의 효소에 대한 비가 계면활성제의 기질에 대한 비보다 더 중요하다는 것을 알 수 있었다. 관찰된 계면활성제의 역할은 계면활성제 존재하에서 이 효소의 반응속도론적 경향을 평가하는데 도움이 된다. Microsomal cholesterol ester hydrolase of rat brain has been solubilized with good yield and stability. The optimal solubilization was achieved by homogenization and high speed centrifugation of microsomes at 10 mg dry weight/ml of Tris-HCl buffer, pH 7.6, containing 0.75% Triton X-100. The solubilized enzyme was more dependent on exogenous lipid for activity than the microsome-bound enzyme, but the optimal concentration of Triton X-100 was greatly reduced. Series of experiments, in with concentrations of Triton X-100, the substrate, and the solubilized enzyme were varied independently, suggested that the activation effect of the detergent depended more on the ratio of detergent to enzyme than on the ratio of detergent to substrate. The observed role of detergent is helpful to evaluate the kinetic behavior of the enzyme in the presence of detergent.

쥐 뇌 Microsome 의 용해된 Cholesterol Ester Hydrolase 의 반응속도론적 경향

최명언 ( Myung Un Choi ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.4

Microsomal cholesterol ester hydrolase of rat brain has been solubilized with good yield and stability. The optimal solubilization was achieved by homogenization and high speed centrifugation of microsomes at 10 ㎎ dry weight/㎖ of Tris-HCl buffer, pH 7.6, containing 0. 75 % Triton X-100. The solubilized enzyme was more dependent on exogenous lipid for activity than the microsome-bound enzyme, but the optimal concentration of Triton X-100 was greatly reduced. Series of experiments, in with concentrations of Triton X-100, the substrate, and the solubilized enzyme were varied independently, suggested that the activation effect of the detergent depended more on the ratio of detergent to enzyme than on the ratio of detergent to substrate. The observed role of detergent is helpful to evaluate the kinetic behavior of the enzyme in the presence of detergent.

쥐 뇌의 Peroxidase 활성도 세포내 및 뇌 부위별 분포

최명언 ( Myung Un Choi ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.4

The peroxidase activity of rat brain was determined by spectrophotometrically using o-dianisidine as substrate. Basic assay conditions were optimized in order to eliminate the pseudoperoxidase activity. The peroxidase activities were then examined in subcellular fractions as well as in six different regions of the brain. It was found that a major part of the activity was associated with particulated fractions. The specific activity of the microsomal enzyme was roughly twice as high as that of the mitochondrial. The regional distribution of the microsomal activity was quite variable. The highest activity was found in the medulla oblongata and lowest in the striatum. The possible role of the brain peroxidase was birefly assessed.

방사선 조사에 의한 쥐 조직의 포스포리파제 D의 활성 변화

최명선(Myung Sun Choi),조양자(Yang Ja Cho),최명언(Myung-Un Choi) 대한방사선종양학회 1997 Radiation Oncology Journal Vol.15 No.3

목 적 : Phospholipase D (PLD)는 phosphatidylcholine을 phosphatidic acid (PA)와 choline으로 가수 분해 시키는 효소이다. 최근 이 효소는 다른 phospholipase들과 유사하게 세포 신호전달과정에 관여하는 것으로 알려져 많은 관심의 대상이 되고 있으며, 아울러 발암과정에 관여하리라는 추측을 하게 하고 있다. 이 실험에서는 쥐를 방사선 조사하여 각 조직에서 올레산-PLD에 미치는 영향을 관찰하였다. 방 법:PLD assay를 위한 반응 혼합물에는 0.1μCi의 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50 mM HEPES buffer(pH 6.5), 10mM CaCl2와 25mM KF 를 함께 넣어주었다. 생성된 PA는 TLC로 분리하여 그 방사능을 측정하였다. 사용된 동물은 암컷 Wistar 쥐로서 코발트 60 원격치료 기기를 이용, 조사범위를 10cm×10cm로하여 분당 선량율 2.7Gy로 방사선 조사선량 10Gy와 25Gy를 조사 하였다 결 과:PLD 활성은 폐조직에서 가장 높았으며 신장, 근육, 뇌, 비장, 골수, 흉선, 간의 순으로 나타났다. 방사선 조사결과 PLD 활성에 변동을 보인 조직은 흉선, 비장, 폐와 골수이며, 특히 흉선과 비장은 PLD의 활성이 각각 2배 이상 증가한 것으로 관찰되었다. 이와는 반대로 골수의 PLD는 30% 이상 감소한 것으로 나타났다. 한편 PLD 활성값이 가장 낮은 간은 방사선 영향을 거의 받지 않는 것처럼 보였다. 결 론:동물전신에 방사선 조사시 PLD가 가장 민감한 영향을 받는 조직은 림프양 기관과 조혈 세포인 것으로 보여 PLD가 이들 조직의 생리기능과 밀접한 관계가 있음을 암시해 주고있다. 더 나아가 방사선 긴장 (radiation stress)이 이들 조직의 세포증식내지 괴사현상연구에 중요한 수단을 제공해 줄 수 있을 것이다. Purpose:Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. Material and Methods :The reaction mixture for the PLD assay contained 0.1μCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer(pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm×10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. Results:Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward γ-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. Conclusion:The PLD activities affected most sensitively by the whole-body

Basic Properties of Brain Monoamine Oxidase Using Benzylamine as Substrate

박희섭,최명언,Park, Hee-Seob,Choi, Myung-Un 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.3

쥐 뇌의 monoamin oxidase(MAO)를 benzylamine을 기질로 하여 분광학적 방법으로 측정했다. 먼저 기본적인 정량조건을 수렵했으며 이 조건하에서 분리된 mitochondria 에서의 MAO 활동도는 2nmoles/분/mg 단백질 정도였다. MAO 의 활동도와 mitochondria의 물리-화학적 상태와의 연관성을 알아 보기 위해 여러 종류의 화학물질들의 영향을 관찰했다. 검토된 중에서 수소와 $Ca^{+2}$ 이온의 영향이 흥미롭게 나타났다. pH가 7.2에서 9.0으로 변하면 MAO의 활동도는 두 갑절이상 증가하며 알카리성일때 계산된 Km 값은 산성일때의 것과 비교 다섯 갑절 정도 적게 나타났다. 따라서 Vmax/Km은 중성분자가 실제로 활성이 있는 기질임을 암시해 주고 있다. pH 7.2와 9.0사이에서 $Ca^{+2}$ 이온은 뇌 MAO의 활동도를 어느 정도 증가시켰으며, 이 효소가 Triton X-100로 용해되었을 경우 이 $Ca^{+2}$ 이온 효과는 없어졌다. Monoamine oxidase (MAO) of rat brain was assayed by spectrophotometric method using benzylamine as substrate. Basic assay condition was optimized and the specific activity of prepared mitochondrial fraction was 2 nmoles/mg protein/min. under the standared condition. In order to correlate the MAO activity with the physico-chemical state of mitochondria, the enzyme activity was assayed in the presence of various chemicals known to affect the metabolic state of mitochondria. Among the tested, the proton and $Ca^{+2}$ ion exibited interesting results. When pH was altered from 7.2 to 9.0, the activity of the brain MAO increased more than twofold and the calculated Km value at the alkaline pH was about 5 times smaller than that of neutral pH. Thus the Vmax/Km profile indicates that the neutral molecule appears to be the active substrate. $Ca^{+2}$ ion showed moderate activation of the brain MAO activity at pH 7.2~9.0 in Tris-HCl buffer. When the mitochondrial MAO was solubilized with Triton X-100, the $Ca^{+2}$ ion effect disappeared.

Xanthomonas citri의 5S rRNA 의 구조 결정

조봉래,최명언,서세원,임자혜,고문주,박인원,Bongrae Cho,Myung-Un Choi,Se Won Seh,ahei Ihm,Moonjoo Koh,Inwon Park 대한화학회 1992 대한화학회지 Vol.36 No.3

Xanthomonas citri의 5S rRNA를 분리, 정제하여 효소적 방법과 화학적 방법으로 그 구조를 결정하였다. 이 5S rRNA는 119개의 누클레오티드로 구성되어 있으며 변형된 누클레오시드를 함유하지 않는다. 그리고 이 5S rRNA는 X. maltophilia의 것처럼 5'-말단에 가외의 우리딘 잔기를 하나 더 가지고 있다. 결정한 X. citri의 5S rRNA의 이차구조는 다른 원핵세포의 것들에 대해서 제안된 일반 모형들과 매우 유사하며 [De Wachter et al., Biochimie, 64, 311 (1982); Specht et al., Nucleic Acids Res., 18, 2215 (1990); Cho et al., Proceedings of the First Symposium on Biomolecules, p. 9 (1991)], 5개의 이중나선 줄기와 5개의 단일가닥 고리 그리고 2개의 내밀린 구조를 가진다. The structure of th 5S rRNA isolated from Xanthomonas citri was determined by enzymatic and chemical degradation methods. It consists of 119 nucleotides and contains no modified nucleosides. As does the 5S rRNA of X. maltophilia, it contains an additional uridine residue on the 5'-terminus. The secondary structure of the 5S rRNA from X. citri was almost identical to the generalized models proposed by many other workers [De Wachter et al., Biochimie, 64, 311 (1982); Specht et al., Nucleic Acids Res., 18, 2215 (1990); Cho et al., Proceedings of the First Symposium on Biomolecules, p. 9 (1991)]. The secondary structure of 5S rRNA from X. citri consists of five helices, five loops and two bulges. This 5S rRNA has a uridine residue at position 66 as a bulge.

쥐 뇌의 용해단백질 황산기 전달효소의 식별과 부분특성 연구

이지오,최명언 ( Jie Oh Lee,Myung Un Choi ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

Protein sulfotransferase (S) in soluble fraction of rat bran were characterized using [^(35)S] phosphoadenosinephosphosulfate (PAPS) as sulfate donor. [^(35)S] PAPS, the universal sulfate donor, was prepared enzymatically in rat liver high speed supernatant with carrier free ^(35)S0₄ and ATP. The activity of soluble protein sulfotransferase was observed in assay system containing [^(35)S] PAPS, Tris-maleate buffer pH 6.7 and soluble proteins from rate brain. The sulfated proteins were separated by SDS-PAGE and acid treatment was performed on the gel to differentiate tyrosine sulfate from carbohydrate sulfate. Incorporation of sulfate into tyrosine residues of proteins of various molecular weights was observed. Particularly high molecular weight ($gt;200 kD) proteins were more intensively sulfated. Effects of incubation time, temperature, pH, amounts of proteins, PAPS concentration, various metal ions, and some polyamino acids on the activities of protein sulfotransferases were investigated. These general properties of the soluble sulfotransferase were mostly distinct from those of the microsome associated sulfotransferase.

벤질아민을 기질로 사용한 뇌 Monoamine Oxidase 의 기초 성질

박희섭,최명언 ( Heeseob Park,Myung Un Choi ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

Monoamine oxidase (MAO) of rat brain was assayed by spectrophotometric method using benzylamine as substrate. Basic assay condition was optimized and the specific activity of prepared mitochondrial fraction was 2 nmoles/㎎ protein/min. under the standared condition. In order to correlate the MAO activity with the physico-chemical state of mitochondria, the enzyme activity was assayed in the presence of various chemicals known to affect the metabolic state of mitochondria. Among the tested, the proton and Ca^(+2) ion exibited interesting results. When pH was altered from 7.2 to 9.0, the activity of the brain MAO increased more than twofold and the calculated Km value at the alkaline pH was about 5 times smaller than that of neutral pH. Thus the Vmax/Km profile indicates that the neutral molecule appears to be the active substrate. Ca^(+2) ion showed moderate activation of the brain MAO activity at pH 7.2×9.0 in Tris-HCl buffer. When the mitochondrial MAO was solubilized with Triton X-100, the Ca^(+2) ion effect disappeared.

지질을 제거한 쥐 뇌의 Monoamine Oxidase 의 용해와 특성 연구

정성권,최명언 ( Sung Kwon Chung,Myung Un Choi ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

In order to investigate the nature of lipid-protein interactions of monoamine oxidase (MAO) of rat brain, the membrane-bound MAO was solubilized with Triton X-100 after depletion of mitochondrial lipid by organic solvents. Extraction with chloroform and acetone removed 9696 of total lipid from mitochondria, while there was no apparent change in enzyme activity. Reactivation was performed by interacting the defined phospholipids with the solubilized MAO. The basic properties of the reactivated enzyme were compared with those of both membrane-bound and solubilized enzyme. It was found that phosphatidylcholine was the activator for the solubilized enzyme with 90% activation at the lipid to protein ratio of 6 to 1. When reactivated the stability of the enzyme improved greatly at 37? and the pH optimum of the solubilized enzyme shifted to that of the membrane-bound enzyme. While K_m value of 0.18 mM of the solubilized enzyme altered to 0.12 mM upon reactivation, the value of V_(max) remained unchanged. It seems that this system could provide a series of basic informations on the roles of phospholipids in rat brain MAO and the nature of the interactions between membrane-bound proteins and their environmental phospholipids.