생쥐 뇌의 세포질 분획에서 Phosphatidylinositol 특이 Phospholipase C의 특성에 대한 연구

김명년,이종호,윤희주,이영식,조기승,Kim, M.N.,Lee, J.H.,Yoon, H.J.,Lee, Y.S.,Cho, K.S. 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3

When the cytosolic phosphatidylinositol-specific phospholipase C (PI-PLase C) activity from mouse brain was estimated by using $H^3$-labelled phosphatidylinositol ($H^3$-PI) as a substrate, it showed the maximum enzyme activity at pH 5.5 with 50 mM Tris-maleate buffer solution. The enzyme activity was increased very sharply at pH 5.5 within 10 min. incubation and slow down after that. But at physiological pH 7.2, the activity was increased continuously until 50 minutes. This enzyme was very sensitive to the reaction temperature and showed a low activity at $0^{\circ}C$ and the maximum hydrolysis occurred at $37^{\circ}C-40^{\circ}C$ When the enzyme activity estimated in the presence of various amounts of enzyme, the maximum activity showed at the addition of $10\;{\mu}l$ ($3.14\;{\mu}g\;protein/{\mu}l$) of enzyme into $200\;{\mu}l$ of reaction volume. In the effects of different kinds of cations, the enzyme was stimulated significantly in the presence of 1 mM calcium ion at the rate of 3.0 times and 4.2 times at pH 5.5. and pH 7.2, respectively. On the contrary, the enzyme activity was inhibited seriously even in the low concentration of $10\;{\mu}M$ mercury and silver ions at both pH. 생쥐 뇌의 세포질 분획에서 $H^3$-phosphatidylinositol ($H^3$-PI)을 기질로하여 phosphatidylinositol-specific phospholipase C (PI-PLase C) 효소 활성도를 보았을 때, 50mM Tris-maleate buffer, pH 5.5인 산성에서 최고의 활성도를 나타냈고, pH 5.5와 pH7.2에서 시간별 활성도를 비교하여 보았을 때 pH 5.5에서는 10분 까지 직선적인 활성도증가를 보였으나 그 이후는 거의 완만한 증가를 나타내는 반면, pH 7.2에서는 50분 까지도 지속척언 활성도 증가를 나타내는 양상을 보였다. 이 효소는 $0^{\circ}C$에서 활성도가 매우 낮았고, $37^{\circ}C-40^{\circ}C$ 사이에서 가장 높은 활성도를 보였으며, 그 이상의 온도에서는 급격한 활성도의 감소를 나타냈다. 본 실험의 반응조건하에서 $10\;{\mu}l$ ($3.14\;{\mu}g\;protein/{\mu}l$)의 효소량이 가장 높은 활성도를 보였으며 효소 단백질의 증가에 따라서 활성도는 오히려 약간의 감소를 나타냈다. 이 효소는 pH 5.5와 pH 7.2에서 여러 cation들의 영향을 보았을 때, 특히 $Ca^{2+}$에 의해서 각각 3.0배와 4.2배의 현저한 활성화 효과를 나타냈으며, $Hg^{2+}$와 $Ag^+$에 의해서는 $10\;{\mu}M$ 농도에서도 심한 저해효과를 나타냈는데, 중성 pH보다 산성 pH에서 더 현저한 저해효과를 나타냈다.

대장균에 있어서 영양물 흡수에 미치는 Palmitoylcarnitine과 인삼 Saponin의 영향

김순옥,이호용,이종삼,최영길,조기승,Kim, Soon-Ok,Lee, Ho-Yong,Lee, Chong-Sam,Choi, Yong-Keel,Cho, Key-Seung 한국미생물학회 1984 미생물학회지 Vol.22 No.3

When enterobacterium, Escherichia coli B was cultivated with normal media in the presence of $0.2{\sim}0.6%$ Palmitoylcarnitine and $0.05{\sim}0.2%$ Ginseng Saponin, maximum population growth of the bacteria was presented 71% and 31%, respectively. Such a result, in vitro test, was concluded from the result that both detergents stimulated $C^{14}$-glucose, $C^{14}$-alanine and $C^{14}$-phosphatidylethanolamine uptake into the membrane of cells. The pre-treatment of cells with different amounts of Palmitoylcarnitine from $0.005{\sim}0.05{\mu}$ moles represented a significant increase of uptake, 33% of $C^{14}$-glucose, 129% of $C^{14}$-alanine and 158% of phosphatidylethanolamine at the concentration of $0.05{\mu}$ moles of Palmitoylcarnitine. On the other hand, the result of $C^{-2}%$ Saponin treatment showed the maximum value of uptake, 17% of $C^{14}$-glucose and 112% of $C^{14}$-alanine. In case of $C^{14}$-phosphatidylethanolamine, the maximum uptake showed 25% of increase at the concentration of $C^{14}$% Saponin.

생쥐 뇌의 세포질 분획에서 Phosphatidylinositol특이 Phospholipase C의 특성에 대한 연구

김명년,이종호,윤희주,이영식,조기승 ( M . N . Kim,J . H . Lee,H . J . Yoon,Y . S . Lee,K . S . Cho ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3

When the cytosolic phosphatidylinositol-specific phospholipase C (PI-PLase C) activity from mouse brain was estimated by using H³-labelled phosphatidylinositol (H³- PI) as a substrate, it showed the maximum enzyme activity at pH 5.5 with 50 mM Tris-maleate buffer solution. The enzyme activity was increased very sharply at pH 5.5 within 10 min. incubation and slow down after that. But at physiological pH 7.2, the activity was increased continuously until 50 minutes. This enzyme was very sensitive to the reaction temperature and showed a low activity at 0℃ and the maximum hydrolysis occurred at 37℃-40℃. When the enzyme activity estimated in the presence of various amounts of enzyme, the maximum activity showed at the addition of 10 ㎕ (3.14 ㎍ protein/㎕) of enzyme into 200 ㎕ of reaction volume. In the effects of different kinds of cations, the enzyme was stimulated significantly in the presence of 1 mM calcium ion at the rate of 3.0 times and 4.2 times at pH 5.5. and pH 7.2, respectively. On the contrary, the enzyme activity was inhibited seriously even in the low concentration of 10 μM mercury and silver ions at both pH.

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.

Structural Characterization of a New Antibiotic Substance Purified from Scolopendra Subspinipes Multilans L. Koch

김기태,김효준,박경배,조기승,Kim, Ki Tae,Kim, Hyo Joon,Park, Kyung Bae,Cho, Key Seung Korean Chemical Society 1998 대한화학회지 Vol.42 No.2

Centipede Scolopendra subspinipes multilans L. KOCH로부터 정제된 항균 물질 Centipedin의 항균 Spectrum 및 작용 Mechanism 연구

김기태,홍사원,원호식,김효준,박경배,조기승,Kim, Ki-Tae,Hong, Sa-Weon,Won, Ho-Shik,Kim, Hyo-Joon,Park, Kyung-Bae,Cho, Key-Seung 한국미생물학회 1998 미생물학회지 Vol.34 No.1

지네 Scolopendra subspinipes multilans L. Koch로부터 정제한 Centipedin의 gram-negative, gram-positive bacteria 및 fungi에 대한 광범위 항균 spectrum을 조사하였으며 액체배지를 사용하여 각 균주에 대한 minimal inhibitory concentrations(MICs)값을 측정하였다. 인체의 기관중 주로 폐에 감염되는 gram-negative bacteria인 Klebsiella pneumoniae ATCC 8308 균주에 대한 MIC값이 $2{\mu}g/ml$로서 가장 강한 항균력을 나타내었고 화농균인 Proteus vulgaris NRRL B-123 균주에 대해서도 MIC값이 $16{\mu}g/ml$로 비교적 높은 항균력을 나타내었으며 항균제로서의 작용 mechanism을 시험한 결과 prokaryotic RNA transcription을 억제함을 알 수 있었다. Human blood leukemia(HL-60)와 mouse B lymphocyte myeloma cell 등의 동물세포에 대한 Centipedin의 cytotoxicity는 없는 것으로 확인되었다. A broad spectrum of antibiotic action was studied with Centipedin purified from centipede Scolopendra subspinipes multilans L. Koch aganist gram-positive, gram-negative bacteria and fungi. The minimal inhibitory concentrations(MICs) were determined in liquid medium. The significant antibiotic activity was obtained aganist gram-negative Klebsiella pneumoniae ATCC 8308 responsible for causing infection at lung and intestine. The MIC value against Klebsiella pneumoniae ATCC 8308 was $2{\mu}g/ml$, and this Centipedin was active against Proteus vulgaris NRRL B-123. In addition, it has been shown that Centipedin blocks procaryotic RNA transcription and a little of DNA replication system in vitro. Centipedin did not exhibit any significant cytotoxicity against animal cells such as human blood leukemia (HL-60) and mouse B lymphocyte myeloma cell.