흰쥐 태반의 Transamidinase 촉매반응에 관여하는 아미노산 잔기의 화학변형

이군자,조영동,Lee, Koon-Ja,Cho, Young-Dong 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.8

흰쥐 태반으로부터 transamidinase를 정제한 후 화학변형의 방법을 이용하여 이 효소의 활성부위에 존재하는 아미노산 잔기를 확인하였다. Transamidinase는 sulfhydryl기와 특이하게 반응하는 N-ethylmaleimide, 5'-dithiobis(2-nitrobenzoate)에 의하여 빠르게 활성이 감소되었으며, N-ethylmaleimide에 의한 비활성 반응은 pseudo first-order kietics로 나타났고 2차 반응속도 상수는 $904 M^{-1}{\cdot}min^{-1}$이었으며 반응차수는 0.8이었다. N-ethylmaleimide에 의한 효소의 비활성은 가역반응으로 dithiothreitol에 의하여 활성이 회복되었으며, 기질인 L-arginine에 의하여 보호효과를 나타내었다. 이 효소는 serine과 특이하게 반응하는 diisopropylflurophosphate에 의해서도 비활성되었으며 이 때의 2차 반응속도 상수는 $74 M^{-1}{\cdot}min^{-1}$, 반응차수는 1.1이었다. DFP에 의한 효소의 비활성은 기질인 L-arginine과 glycine에 의하여 모두 보호효과를 나타내었다. 이러한 결과로부터 cysteine과 serine 잔기가 각각 1개씩 이 효소의 활성에 관여할 것으로 추정된다. We examined the chemical modification of the purified rat placenta transamidinase. The enzyme was inactivated by sulfhydryl group specific modification reagents such as NEM and DTNB. The enzyme was turned out to be inactivated by NEM rapidly and the reaction followed pseudo first-order kinetics. The second-order rate constant and reaction order with respect with NEM were $904 M^{-1}{\cdot}min^{-1}$ and 0.8, respectively. Inactivation by the NEM was restored to almost orginal activity by DTT and was protected by the L-arginine. The transamidiase was also inactivated by the DFP. The second-order rate constant and the reaction order with respect to DFP were $74 M^{-1}{\cdot}min^{-1}$ and 1.1, respectively. L-arginine and glycine provided almost complete protection against inactivation by DFP. Such cumulative results suggest that cysteine and serine residues seems to be involved in enzyme activity.

Mouse 신장으로부터 Ornithine decarboxylase 의 정제

이군자,조영동 ( Koon Ja Lee,Young Dong Cho ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.5

Ornithine decarbosylase (ODC) was purified to homogenity from testosterone treated mice kidney by ammonium sulfate fractionation, DEAE-Sephacel chromatography, hydroxylapatite chromatography, pyridoxamine-5-phosphate-agarose affinity chromatography. The specific activity of ODC was 14,000 U/㎎, and purification fold was 1,180. The enzyme has a Mr. of about 110,000 dalton and is a dimer of subunit Mr. 52,000. The K_m for L-ornithine was 167 μM and for pyridoxamine phosphate, 142 nM. ODC has an optimum pH of 7.0 and optimum temperature of 37℃. The purified enzyme was not inhibited by polyamine (putrescine, spermidine and spermine). During purification process, ODC stabilizing material was found.

흰쥐 태반의 Transamidinase 촉매반응에 관여하는 아미노산 잔기의 화학변형

이군자,조영동 ( Koon Ja Lee,Young Dong Cho ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

We examined the chemical modification of the purified rat placenta transamidinase. The enzyme was inactivated by sulfhydryl group specific modification reagents such as NEM and DTNB. The enzyme was turned out to be inactivated by NEM rapidly and the reaction followed pseudo first-order kinetics. The second-order rate constant and reaction order with respect with NEM were 904M^(-1)·min^(-1) and 0.8, respectively. Inactivation by the NEM was restored to almost orginal activity by DTT and was protected by the L-arginine. The transamidiase was also inactivated by the DFP. The second-order rate constant and the reaction order with respect to DFP were 74 M^(-1)·min^(-1) and 1.1, respectively. L-arginine and glycine provided almost complete protection against inactivation by DFP. Such cumulative results suggest that cysteine and serine residues seems to be involved in enzyme activity.

A Study on the Neoasozine Residues in Rice Grain by Neutron Activation Method

Yong-Hwa Kim(金容華),Koon-Ja Lee(李君子),Su-Rae Lee(李瑞來) 한국식품과학회 1981 한국식품과학회지 Vol.13 No.1

흰쥐 태반의 Transamidinase 촉매반응에 관여하는 아미노산 잔기의 화학변형

이군자,조영동 한국생화학회 1993 BMB Reports Vol.26 No.8

흰쥐 태반으로부터 transamidinase를 정제한 후 화학변형의 방법을 이용하여 이 효소의 활성부위에 존재하는 아미노산 잔기를 확인하였다. Transamidinase는 sulfhydryl기와 특이하게 반응하는 N-ethylmaleimide, 5'-dithiobis(2-nitrobenzoate)에 의하여 빠르게 활성이 감소되었으며, N-ethylmaleimide에 의한 비활성 반응은 pseudo first-order kinetics로 나타났고 2차 반응속도 상수는 904M^(-1)·min^(-1) 이였으며 반응차수는 0.8이었다. N-ethylmaleimide에 의한 효소의 비활성은 가역반응으로 dithiothreitol에 의하여 활성이 회복되었으며, 기질인 L-arginine에 의하여 보호효과를 나타내었다. 이 효소는 serine과 특이하게 반응하는 diisopropylflurophosphate에 의해서도 비활성되었으며 이 때의 2차 반응속도 상수는 74M^(-l)·min^(-1), 반응차수는 1.1이었다. DFP에 의한 효소의 비활성은 기질인 L-arginine과 glycine에 의하여 모두 보호효과를 나타내었다. 이러한 결과로부터 cysteine과 serine 잔기가 각각 1개씩 이 효소의 활성에 관여할 것으로 추정된다. We examined the chemical modification of the purified rat placenta transamidinase. The enzyme was inactivated by sulfhydryl group specific modification reagents such as NEM and DTNB. The enzyme was turned out to be inactivated by NEM rapidly and the reaction followed pseudo first-order kinetics. The second-order rate constant and reaction order with respect with NEM were 904M^(-1)·min^(-1) and 0.8, respectively. Inactivation by the NEM was restored to almost orginal activity by DTT and was protected by the L-arginine. The transamidiase was also inactivated by the DFP. The second-order rate constant and the reaction order with respect to DFP were 74 M^(-1)·min^(-1) and 1.1, respectively. L-arginine and glycine provided almost complete protection against inactivation by DFP. Such cumulative results suggest that cysteine and serine residues seems to be involved in enzyme activity.

Mouse 신장으로부터 Ornithine Decarboxylase의 정제

이군자,조영동 한국생화학회 1992 BMB Reports Vol.25 No.5

자성 mouse의 피하층에 testosterone을 투여하여 ornithine decarboxylase의 활성을 증가시킨 후, 신장으로부터 ornithine decarboxylase를 황산암모늄 분별침전, DEAE-sephacel chromatography, hydroxylapatite chromatography, pyridoxamine-5'-phosphate agarose affinity chromatography를 이용하여 분리 정제하였다. 이 효소의 분자량은 SDS-polyacrylamide gel electrophoresis, Sephacryl S-200 gel chromatography와 ornithine decarboxylase의 효소활성 억제제인 14C-DFMO 을 효소와 결합시켜 얻은 autoradiography를 이용하여 52,000 dalton인 동일한 subunit으로 구성된 dimer임을 알 수 있었다. 정제된 효소의 ornithine에 대한 K_(m) 값과 V_(max) 값은 각각 167㎛과 5 nmol CO_(2)/h로 나타났고, pyridoxal-5-phosphate에 대한 K_(m) 값과 V_(max) 값은 각각 0.142㎛과 2.4nmol CO_(2)/h로 나타났다. 효소의 최적 pH 는 7.0, 최적 온도는 37℃이었으며, 정제된 ornithine decarboxylase는 polyamines(putrescine, spermidine, spermine)에 의해 효소 활성이 영향을 받지 않았다. 본 실험에서는 ornithine decarboxylase의 분리과정 중에서 ornithine decarboxylase 활성을 안정화시키는 물질을 발견하였다. Ornithine decarbosylase (ODC) was purified to homogenity from testosterone treated mice kidney by ammonium sulfate fractionation, DEAE-Sephacel chromatography, hydroxylapatite chromatography, pyridoxamine-5-phosphate-agarose affinity chromatography. The specific activity of ODC was 14,000 U/㎎, and purification fold was 1,180. The enzyme has a Mr. of about 110,000 dalton and is a dimer of subunit Mr. 52,000. The K_(m), for L-ornithine was 167㎛ and for pyridoxamine phosphate, 142 nM. ODC has an optimum pH of 7.0 and optimum temperature of 37℃. The purified enzyme was not inhibited by polyamine (putrescine, spermidine and spermine). During purification process, ODC stabilizing material was found.

태생기 및 신생기 흰쥐 간장에서의 Putrescine 대사에 관한 연구

이군자 서울保健大學 1992 論文集 Vol.12 No.1

Mitomycin C투여에 의한 공막의 조직병리학적 연구

이군자 서울保健大學 1996 論文集 Vol.16 No.1

익상편 수술 후 재발 방지를 위하여 사용되고 있는 MMC를 안구의 성장이 활발한 어린 흰쥐에게 점안한 후 나타나는 공막의 조직학적 및 조작 화학적인 변화를 관찰하여 다음과 같은 결과를 얻었다. 1) 저농도(0.04mg/mL)의 MMC를 점안한 공막에서는 조직학적인 변화를 관찰할 수 없었으나, 고농도(0.1mg/mL) 의 MMC를 3일간 점안한 실험군과 7일간 점안한 실험군에서는 공막실질의 아교섬유의 간격이 상당히 벌어졌으며 이러한 조직 손상은 MMC 점안을 중지한 후에도 완전히 회복되지 않았다. 2) 고농도(0.lmg/mL)의 MMC를 점안한 실험군의 공막실질에서 alkaline phosphatase 활성은 약간 감소하였으며, MMC 투여 후 7일이 경과한 경우에도 효소활성이 회복되지 않았다. 3) 저농도(0.04mg/mL) 및 고농도(0.lmg/mL)는 MMC를 점안한 실험군의 공막실질에서 섬유모세포와 제I형 아교질 항체에 대한 반응은 대조군에 비하여 감소하였으며, MMC 점안 후 7일이 경과한 실험군에서도 제I형 아교질항체에 대한 반응은 회복되지 않았다. 4) 저농도의 MMC를 1회 점안한 실험군, 3일간 점안한 실험군, 접안 후 7일 경과군 및 고농도의 MMC를 1회 투여한 실험군의 공막실질에서는 Cu, Zn-SOD 활성이 관찰되었으나, 고농도의 MMC를 장기간 점안한 실험군과 점안 후 7일이 경과한 실험군에서는 활성이 관찰되지 않았다. 이상과 같은 결과로 MMC를 점안한 흰쥐의 공막실질에서는 일시적으로 Cu, Zn-SOD 의 활성이 증가하여 MMC에 의한 조직독성을 일부 줄일 수 있을 것으로 생각되나, MMC를 계속 점안한 경우에는 alkaline phosphatase 활성이 감소하고 아교섬유의 합성능력이 감소하여 공막실질의 아교섬유가 정상적인 판(lamella) 을 형성하지 못하여 공막괴사 등의 부작용이 나타나는 것으로 생각되며, 고농도의 MMC를 점안한 경우에는 점안을 중지한 후에도 공막에 조직독성이 나타나는 것으로 생각된다. Mitomycin C(MMC), widely used as an adjunct in the surgical treatment of pterygium, was instilled to the young rats to evaluate the histologic and histopathologic toxicity of the sclera. 1. Significant histologic changes were not detected in the lower dose of MMC(0.04mg/mL) treated groups, but lamella structure of sclera was irregular in the higher dose of MMC(0.1mg/ mL) treated groups. These structural changes were not recovered within 7days after ceasation of MMC instillation. 2. Alkaline phosphatase activity was decreased in the higher dose of MMC treated groups, and it was not recovered within 7 days after ceasation of MMC instillation. 3. Type I collagen and type I collagen synthetic activity of fibroblast were decreased in the lower and higher dose of MMC treated groups, and these changes were not recovered within 7 days after ceasation of MMC instillation. 4. Cu, Zn-SOD activity was increased in the lower dose of MMC treated groups and single application of higher dose of MMC, but Cu, Zn-SOD activity was not detected in the other higher dose of MMC treated groups. These results indicate that increase of Cu, Zn-SOD activity may reduce the MMC toxicity temporarily, but repeated MMC treatment changes the sclera irregular by decreasing biosynthesis of collagen in the fibroblast. These irregular lamella structure may lead to necrotizing scleritis.

Dichlorvos가 mouse간장의 인산염분해효소 활성에 미치는 영향

이규식,김원경,이군자,정호삼 漢陽大學校環境科學硏究所 1987 環境科學論文集 Vol.8 No.-

Dichlorvos(DDVP)는 유기인제 화합물로서 체내에서 쉽게 가수분해되어 인체에 대한 독성은 비교적 약하나 농촌에서 살충제와 곤충의 구충제로 사용될 뿐만 아니라 도시에서도 가정용 살충제로 널리 사용되고 있어 그 취급과 부주의로 인하여 급성중독증상이 빈번히 발생되어 사회문제로 대두되고 있다. 다량의 DDVP에 중독되면 carboxyl esterase활성이 억제되어 acetylcholine이 축적되고 이로 인하여 신경기능장애가 나타나며 cyclic-AMP의 분비가 증가되어 탄수화물, 단백질 및 지방의 대사과정에도 변화가 나타나게 된다. 이에 저자는 DDVP를 실험동물에 투여하면 이로 인한 신경기능장애를 일으킬 뿐 아니라 간장에서 해독과정을 거치면서 간실질세포에 독성이 나타나리라 사료되어 alkaline phosphatase와 adenosine triphosphatase활성을 조직화학적으로 검색하였다. 실험동물로는 체중 20gm내외의 ICR계 웅성 mouse에 체중 kg당 40mg의 DDVP를 주사용 증류수 0.2ml에 희석하여 복강내로 주사하고 1.5시간, 3시간, 6시간, 12시간 및 24시간 경과 후에 희생시켜 간장의 좌측전엽을 적출하고 alkaline phosphatase활성은 Gomori법으로, adenosine triphosphatase활성은 Wachstein-Meisel법으로 염색하여 다음과 같은 결과를 얻었다. 1. Alkaline phosphatase활성은 DDVP투여 1.5시간 경과군에서 mouse간장의 간소엽 주변대와 중간대에서 강한 양성반응을 나타내어 일시적인 활성의 증가를 나타내었으나 시간이 경과함에 따라 효소의 활성이 감소하여 DDVP투여 12시간 경과군에서는 간소엽 주변대에서는 중등도의 양성반응을 나타내었으며 간소엽 중간대에서는 음성반응을 나타내었다. 2. Adenosine triphosphatase활성은 DDVP투여 1.5시간 경과군에서는 mouse간장의 간소엽 주변대와 중간대에서 모두 중등도의 양성반응을 나타내었고 DDVP투여 3시간 경과군에서는 간소엽 전역에 걸쳐 강한 양성반응을 나타내었으며 시간이 경과함에 따라 점차 감소하여 12시간 경과군에서는 간소엽 주변대와 중간대에서 약한 양성반응을 나타내고 간소엽 중심대에서는 미약한 양성반응을 나타내었다. 이상의 소견을 종합한 바 DDVP는 mouse간장에서 초기단계에서는 물질대사에 관여하는 alkaline phosphatase및 adenosine triphosphatase활성이 증가되나 시간이 경과함에 따라 이들 효소활성이 저하되는 것으로 사료된다. Dichlorvos(DDVP) as an organophosphorus compound is so easily and rapidly hydrolyzed that it has a little toxic effect on man. But it has been so widely used as an industrial and domestic insecticide and anthelmintic agent for animals that the accident of chemical poisoning occurs frequently. DDVP is a powerful inhibitor of carboxylic esterase resulting in accumulation of acetylcholine at the synapses, which in turn causes paralysis of the transmission in cholinergic synapses. And the accumulation of the acetylcholine brings the elevation of the cyclic-AMP level that alters the metabolism of carbohydrate, protein and lipid. Owing to changes in the metabolism and the detoxification in the liver, hepatic parenchymal cells may be damaged. Therefore, the author undertook the present study to pursue the toxic effect of the DDVP on the phosphatase activity in the liver. Albino mice, ICR strain, weighing 20gm were used as experimental animals. The experimental animals sacrificed at 1.5,3,6,12 and 24 hours after administration of 40mg/kg of DDVP. The specimens obtained from the liver were sectioned with 16㎛ thickness in a frozen cryostat. The activity of alkaline phosphatase was observed by the Gomori's method and the activity of adenosine triphosphatase was observed by the Wachstein-Meisel's method for histochemical study. The results obtained are as follows. 1. The activity of alkaline phosphatase was strongly positive in the periportal and central zones of the hepatic lobule of the 1.5 hour-DDVP treated group. The activity of alkaline phosphatase was decreased with time, and moderate positive activity in the periportal zone and negative activity in the central and intermediate zones of the hepatic lobule were observed in the 12 hour-DDVP treated group. 2. The activity of adenosine triphosphatase was moderately positive in the periportal and central zones of the hepatic lobule of the 1.5 hour- DDVP treated group and the strongly positive in the entire hepatic lobule of the 3 hour-DDVP treated group. The activity of adenosine triphosphatase was decreased with time and weak positive activity in the periportal and intermediate zones and trace positive activity in the central zone of the hepatic lobule were observed in the 12 hour- DDVP treated group.

Vincristine 이 Mouse 간장의 인산염분해효소 활성에 미치는 영향

이군자,김자영,이규식 한양대학교 의과대학 1987 한양의대 학술지 Vol.7 No.2

Vincristine is a cytotoxic tubulin binding agent, derived from the periwinkle plant, Vinca rosea Linn. It has been known as a potent mitotic inhibitor that has found their place as effective drug in combination chemotherapy regimens for the treatment of humanleukemias, lymphomas and a variety of solid tumors. The major antitumor effect of this agent appears to be related to its high affinity binding to the basic protein subunit of microtubules, tubulin, which results in disruption of the mitotic spindle apparatus and arrest cells in metaphase. Neurotoxicity is dose-limiting toxicity of administration of vincristine. Vincristine also acts on normally proliferating cells. The author has investigated the effect of vincristine on the mouse liver histochemially observing the change in the activities of alkaline phosphatase and adenosine triphosphatase (ATPase). The mice of the vincristine treated group were given 2.5mg per kg of body weight of mouse in the form of vincristine in 0.9% sodium chlorid and the animals of control group were given 0.9% sodium chlorid only through intraperitoneal injection. After administration, the animals were killed at intervals of 6, 12, 24 and 36 hours. The specimens which were obtained from the anterior lobe or of the liver, were fixed in 10%-neutral formalin at 4℃ and sectioned at 16㎛ thickness in frozen cryostat. He activities of alkaline phosphatase and ATPase were observed by Gomori's method and by Wachstein-Meisel's method, respectively. The results are as follows: 1. The activity of alkaline phosphatase was weakly positive in the perioportal and intermedicate zones, trace positive in the central zone of the hepatic lobule of the 6 hours vincristine treated group. Trace positive reaction was observed in the hepatic lobule of 12 hours vincristine treated group. Negative reaction in the central zone, trace positive reaction in the intermediate zone and weakly positive reaction in the periportal zone were observed. Trace positive reaction was observed in the central zone and weakly positive reaction was seen in the intermediate and periportal zone of the hepatic was seen in the intermediate and periportal zone of the hepatic lobule of 36 hours vincristine treated group. 2. ATPase activity was weakly positive in the central and periportal zones and trace positive in the intermediate zone of the hepatic lobule of 6 hours vincristine treated group. Trace positive reaction was observed in the entire hepatic lobule of 12 hours vincristine treated group. There are weakly positive in the 24 hours vincristine treated group and moderately positive reaction in the whole hepatic lobule of the 36 hours vincristine treated group. Consequently, it is suggested that vincristine decreases activities of alkaline phosphatase and ATPase in the liver, due probably to the cytotoxic effect of the drug on the liver.