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Calcium ion과 ATP에 의해 기질선택성이 조절될 수 있는 Phosphatidylinositol-specific phospholipase C에 대한 연구
황재택,이종호,이영식,이서구,조기승,Hwang, J.T.,Lee, J.H.,Lee, Y.S.,Rhee, S.G.,Cho, K.S. 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
생쥐 뇌의 세포질 분획에서 추출한 Phosphatidylinositol-Specific Phospholipase C (PI-PLC, EC 3.1.4.16)를 $^3H$-Phosphatidylinositol ($^3H-PI$)과 $^3H$-Phosphatidylinositol-4.5-bisphosphate ($^3H-PIP_2$)를 기질로 하여 최적 pH 5.0과 생리적 pH 7.2에서 활성도를 보았을 때, 이 효소의 기질선택성이 $Ca^{2+}$ 농도와 ATP 농도에 의해 조절될 수 있음을 나타냈다. $Ca^{2+}$이 존재하지 않은 상태의 pH 5.0과 pH 7.2에서 $PIP_2$는 PI보다 각각 약 4배와 8배의 가수분해도를 나타냈고, 이 조건에서 PI 가수분해는 매우 낮았다. $Ca^{2+}$ 농도를 증가시켰을 때 PI의 가수분해도 pH 5.0과 pH 7.2에서 모두 5 mM까지 계속 증가하는 양상을 보였으나, $PIP_2$ 기질은 pH 5.0에서 $Ca^{2+}$ 농도 1 mM까지 약간 증가하다가 그 이상의 농도에서는 감소현상을 나타냈으며, pH 7.2에서는 0.2 mM까지 증가하다가 그 이상의 농도에서는 급격히 감소되는 결과를 나타냈다. 생리적 pH 7.2에서 ATP에 대한 영향을 보았을 때, PI 기질은 $Ca^{2+}$이 존재하지 않은 경우 아무런 영향이 없었으나 1mM $Ca^{2+}$ 존재하에서는 ATP $10^{-5}M$ 이상의 농도에서 급격히 감소하였다. 한편 $PIP_2$ 기질의 경우, $Ca^{2+}$이 존재 하지 않은 상태에서는 $10^{-3}M$ ATP에 의해 약 100%의 가수분해 증가를 나타냈고, 1 mM $Ca^{2+}$ 존재하에서는 $10^{-2}M$ ATP에 의해 약 150 %의 증가를 나타냈다. Previously reported mouse brain cytosolic PI-phospholipase C (Kim et al., 1987) showed the selectivity of substrates, such as, $^3H-PI$ and $^3H-PIP_2$ was possibly regulated by the concentration of $Ca^{2+}$ and ATP at the optimum pH 5.0 and physiological pH 7.2. The enzymatic hydrolysis of $PIP_2$ at pH 5.0 and pH 7.2 without $Ca^{2+}$ represented about 4 and 8 times higher than PI, respectively. In this condition, PI showed very low hydrolysis. As increase the $Ca^{2+}$ concentrations, the hydrolysis of PI at both pH 5.0 and pH 7.2 showed steady increase until 5 mM $Ca^{2+}$ concentration. On the other hand, the hydrolysis of $PIP_2$ at pH 5.0 showed a little increase until 1 mM $Ca^{2+}$ concentration and decreased after that. And also, the similar result was obtained at pH 7.2 with 0.2 mM $Ca^{2+}$ concentration. In the effect of A TP on the hydrolysis of PI and $PIP_2$ at the physiological pH 7.2, PI hydrolysis was not affected at all without $Ca^{2+}$, but it was significantly decreased over $10^{-5}M$ATP concentration with 1 mM $Ca^{2+}$. In the case of $PIP_2$ ATP showed 100% stimulation with $10^{-3}$M ATP in the absence of $Ca^{2+}$, and 150 % with $10^{-2}M ATP in the presence of 1 mM $Ca^{2+}$, respectively.
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Calcium ion과 ATP 에 의해 기질선택성이 조절될 수 있는 Phosphatidylinositol - specific phospholipase C 에 대한 연구
황재택,이종호,이영식,이서구,조기승 ( J . T . Hwang,J . H . Lee,Y . S . Lee,S . G . Rhee,K . S . Cho ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
Previously reported mouse brain cytosolic PI-phospholipase C (Kim et at., 1987) showed the selectivity of substrates, such as, ³H-PI and ³H-PIP₂ was possibly regulated by the concentration of Ca^(2+) and ATP at the optimum pH 5.0 and physiological pH 7.2. The enzymatic hydrolysis of PIP₂ at pH 5.0 and pH 7.2 without Ca^(2+) represented about 4 and 8 times higher than PI, respectively. In this condition, PI showed very low hydrolysis. As increase the Ca^(2+) concentrations, the hydrolysis of PI at both pH 5.0 and pH 7.2 showed steady increase until 5 mM Ca^(2+) concentration. On the other hand, the hydrolysis of PIP₂ at pH 5.0 showed a little increase until 1 mM Ca^(2+) concentration and decreased after that. And also, the similar result was obtained at pH 7.2 with 0.2 mM Ca^(2+) concentration. In the effect of ATP on the hydrolysis of PI and PIP₂ at the physiological pH 7.2, PI hydrolysis was not affected at all without Ca^(2+), but it was significantly decreased over 10^(-5)M ATP concentration with 1 mM Ca^(2+). In the case of PIP₂ATP showed 100% stimulation with 10^(-3)M ATP in the absence of Ca^(2+), and 150 % with 10^(-2)M ATP in the presence of 1 mM Ca^(2+), respectively.
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Hwang, Jae-Taeck,Choi, Dong Wik 慶北大學校 醫科大學 1980 慶北醫大誌 Vol.21 No.2
琴湖江에서 採集되는 淡水魚에서의 吸蟲類 被襄幼蟲의 奇生上의 變化를 알아 보기 위해 1979年 3月부터 1980年 9月까지 同江流域의 4個處 東村, 淸線, 琴湖 및 永川에서 投網, 낚시 및 사발 모지 等으로 淡水魚를 採集하여 吸蟲類 被襄幼蟲의 魚種別 奇生率과 그 奇生程度를 調査한 다음 李(1968)의 調査成績과 比較하였다. 採集된 淡水魚는 큰납지리, 칼납자루, 붕어, 꼬치동자개, 꺽지, 몰개, 누치, 버들붕어, 납지리, 모래모치, 참붕어, 돌고기 및 피래미의 13種이었고 이들 淡水魚에서 7種의 吸蟲類 被襄幼蟲 Clonoechis sinensis, Cyathocotyle orientalis, Echinochasmus species, Exorchis oviformis, Metacercaria hasegawai, Meta onimus yokogawai, 및 Metrochis orientalis와 3種의 所屬不定 吸蟲類 被襄幼蟲 A, B, 및 C를 魚肉에서 찾아볼 수 있었다. Exorchis oviformis幼蟲은 13種의 全魚類에서 찾아볼 수 있었는데 비하여 Metacercaria hasegawai幼蟲은 12種, Clonorchis sinensis幼蟲은 11種, Cyathocotyle orientalis幼蟲은 9種, Metagonimus yokogawai幼蟲은 8種, Echinochasmus species는 6種 및 Metrochis orientalis는 4種의 魚類에서 찾아볼 수 있었다. 肝吸蟲 被襄幼蟲의 奇生率은 큰납지리, 칼납자루, 몰개, 및 납지리에서는 1968年의 그率에 比해 높았는데 反하여 누치와 모래모치에서는 오히려 떨어졌고 참붕어와 돌고기에서는 奇生率의 變動을 찾아 볼 수 없었다. 肝吸蟲 被襄幼蟲의 奇生程度의 標識로서 魚肉 1 gram當 被襄幼蟲數는 칼납자루와 돌고기에서는 1968年에 比해 이번 調査에서는 增加되었는데 反하여 누치와 납지리에서는 減少되었고 큰납지리, 몰개, 모래모치 및 참붕어에서는 有意的差를 認定할 수 없었으며 例外없이 참붕어에서 肝吸蟲 被襄幼蟲의 奇生數가 가장 많았다. 肝吸蟲을 除外한 吸蟲類 被襄幼蟲의 魚肉內 奇生率과 그 奇生程度의 比較는 3種의 被襄幼蟲 즉 Cyathocotyle orientalis, Exorchis oviformis 및 Metacercaria hasegawai에서만 可能하였는데 大體로 이번 調査에서는 1968年의 그率과 程度에 比해 모두 높았다. 비늘 및 지느러미와 꼬리에서의 吸蟲類 被襄幼蟲의 奇生率은 너무 變動이 甚하여 1968年의 그率과 比較할 수 없었다. In order to determine the recent patterns of infestation with the encysted larvae of digenetic trematodes from fresh-water fish collected in the river Kumho in Kyungpook Province, Korea, fish caught in the 4 localities, Dong-chon, Cheong-cheon, Kumho, and Yeong-cheon of the river were examined from March, 1979 to September, 1980. The fish were collected using following techniques; fishing, throwing a castnet, and using a bait of crushed oil cake in a transparent plastic bowl. The results were compared with the data obtained from fresh-water fish caught at the same river by Lee (1968). Thirteen species of fish, the Korean rose bitterling, Achanthorhodeus taenianalis, the oily bitterling, Acheilognathus limbata, the crussian carp, Carassius carassius, the bullhead, Coreobagrus brevicorpus, the perch, Coreoperca herzi, the Korean shiner. Gnathopogon atromaculatus, the Korean barbel, Hemibarbus labeo, the round tailed fighting fish, Macropodus chinensis, the flat bitterling, Paracheilognathus rhombea, the gudgeon, Pseudogobio esocinus, the southern top-mouthed minnow, Pseudorasbora parva, the striped shiner, Pungtungia herzi, and the pale chub, Zacco platypus, were examined for the presence of the encysted larvae of digenetic trematodes. Seven species of the metacercariae, Clonorchis sinensis, Cyathocotyle orientalis, Echinochasmus species, Exorchis oviformis, Metacercaria hasegawai, Metagonimus yokogawai, and Metrochis orientalis, and three kinds of undetermined cyst A, B, and C were found. Of these, the encysted larva of Exorchis oviformis was found most frequently from all 13 species of fresh-water fish, followed by the cyst of Metacercaria hasegawai from 12 species, the metacercaria of Clonorchis sinensis from 11 species, Cyathocotyle orientalis from 8 species, Echinochasmus species from 6 species, and Metorchis orientalis from 4 species of fish. The infestation rates for the metacercaria of Clenoerchis sinensis in Acanthorhodeus taenianalis, Acheilognathus limbata, Gnathopogon atromaculatus, and Paracheilognathus rhombea in the present study (1980) were higher than those reported by Lee(1968). By contrast the rates in Hemibarbus labeo and Psedegobio esocinus decreased, while no variation of the rates in Pseudorasbora parva and Pungtungia herzi was encountered. In the intensity of infestation, the average number of the cyst of Clonorchis sinensis per gram of flesh in Acheilognathus limbata, and Pungtungia herzi in 1980 increased compared with the average in 1968. By contrast, the number decreased in Hemibarbus labeo and Paracheilognathus rhombea, but no fluctuation in the intensity of infestation in Acanthorhodeus taenianalis, Gnathopogon atromaculatus, Pseudogobio esocinus, and Pseudorasbora parva was observed. In the both years surveyed, Pseudorasbora parva was the most heavily infested with the cyst of Clonorchis sinensis. The comparative data for the infestation rate and the intensity of infestation of the encysted larvae of Cyathocotyle orientalis, Exorchis oviformis, and Metacercaria hasegewai were obtained in both 1968 and 1980 surveys. The rates and intensities in the cysts of the three larvae in the present study increased compared with those in 1980. However, it was impossible to compare the infestation rates for the encysted larvae in the scales, fins and tail as they varied so considerably in both 1968 and 1980 surveys.
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생쥐 뇌조직에서 부분정제한 Phosphatidylinositol - Specific Phospholipase C Ⅰ과 Ⅱ의 특성
이종호,황재택,김명년,이영식,조기승 ( J . H . Lee,J . T . Hwang,M . N . Kim,Y . S . Lee,K . S . Cho ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3
Two forms of phosphatidylinositol-specific phospholipase C (PLC-I and PLC-II) were partially purified from mouse brain cytosolic fraction. These enzymes showed optimum pH 5.0 with PI and pH 5.2 with PIP₂ as substrates, respectively. The enzymatic hydrolysis of PI by both isozymes at pH 5.0 and pH 7.2 showed significant increase with increased concentration of Ca^(2+). On the other hand, the hydrolysis of PIP₂ by PLC-I and PLC-II at both pH exhibited the maximum activity in the presence of 0.5 mM and 0.2 mM Ca^(2+), respectively, while inhibited significantly by increasing the concentration of Ca^(2+). Especially without Ca^(2+) at pH 7.2, PIP₂ hydrolysis by PLC-I and PLC-II represented 15 and 4 times higher than those of PI substrate, respectively. From these results, it could be concluded that PIP₂ hydrolysis Ca^(2+) -independent under the same conditions. In the effect of nucleotides on PI hydrolysis at pH 5.0, ATP and GTP inhibited the enzyme activity of both enzymes, but PIP₂ hydrolysis at physiological pH 7.2 by PLC-I and PLC-II enhenced significantly without Ca^(2+). At this condition, 1 mM Ca^(2+) showed no effect at all on the enzyme activity. The results of complete inhibition of the enzyme activity by Ag^+ and Hg^(2+) suggested that -SH group was involved in the active site of both isozymes.
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