대장균 Strain B 와 B / r 에 있어서 Acyl Phosphatidyl Glycerol 의 생합성 기작에 관한 연구

조기승,홍승덕,장정순,이강석 ( Key Seung Cho,Seung Duk Hong,Chung Soon Chang,Kang Suk Lee ) 생화학분자생물학회 1975 BMB Reports Vol.8 No.1

The present study has shown that phosphatidyl glycerol was converted to 3-sn-phosphatidyl-1`-(3`-acyl)-sn-glycerol(acyl phosphatidyl glycerol) by cell free extracts of Escherichia coli B and B/r. In this process, phosphatidyl glycerol was esterified directly rather than degraded to simpler compounds which are subsequently incorporated into acyl phosphatidyl glycerol. The enzyme, $quot;acyl phosphatidyl glycerol synthetase$quot;, which catalyzes the conversion of phosphatidyl glycerol to acyl phosphatidyl glycerol was found to reside solely in the praticulate fraction of E. coli homogenates. Two separate reactions were identified where one molecule of acyl phosphatidyl glycerol was synthesized from two molecules of phosphatidyl glycerol, in which phosphatidyl glycerol acted both as an acyl acceptor and a donor. In the other reaction, phosphatidyl ethanolamine appears to act as an acyl donor to synthesize acyl phosphatidyl glycerol. The formation of acyl phosphatidyl glycerol has an optimum pH value at pH 7.0 and calcium concentration of 6.0 mM. These results show a new pathway for the turnover of phosphatidyl glycerol in Escherichia coli.

사람의 적혈구막의 Palmitoylcarnitine 과 Palmitoyl - Coa Carnitine Palmitine Palmitoyltransferase

조기승,정진성 ( Key Seung Cho,Jin Sung Chung ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.3

An enzyme, palmitoyl-CoA: carnitine palmitoyltransferase (E.C. 2. 3.1.21) was confirmed to catalyze reversively the formation and hydrolysis of ^(14)C-palmitoylcarnitine in human erythrocyte membrane. The optimum pH range of this enzyme was between from pH 7.2 to pH 7.4, and decreased rapidly outside of this pH range. In the several buffer systems tested, the highest enzyme activity was obtained with Tris-HCl buffer. As a cofactor, 5 μmoles of Mg^(2+) ion significantly stimulated the enzyme activity in the experimental conditions. Palmitoylcarnitine formed from this enzyme system or synthesized from other chemical method has activated significantly the membrane bound enzymes, such as adenosine triphosphatase (ATPase) and Glucose-6-Phosphatase (G-6-Pase).

The Mechanism of Acyl Phosphatidyl Glycerol Biosynthesis in Escherichia coli B and B/r

조기승,홍승덕,장정순,이강석,Cho, Key-Seung,Hong, Seung-Duk,Chang, Chung-Soon,Lee, Kang-Suk 생화학분자생물학회 1975 한국생화학회지 Vol.8 No.1

본 연구는 대장균 strain B와 B/r(Escherichia coli Band B/r)의 세포막 분획에의 해서 phosphatidyl glycerol이 3-sn-phosphatidyl-1'-(3'-acyl(-sn-glycerol(acyl phosphatidyl glycerol 로 전환되는 기작을 밝혔다. 이 과정에서 phosphatidyl glycerol은 더 간단한 화합물로 분해된 후 acyl phosphatidyl glycerol로 합성되는 것이 아니라 직접 acyl group이 옮겨져서 ester 결합을 하는 것으로 생각된다. Phosphatidyl glycerol이 acyl phosphatidyl glycerol로 전환되는 반응을 촉매하는 효소인 acyl phosphatidyl glycerol synthetase는 E. coli homogenate의 세포막 분획에 존재하는 것으로 밝혀졌다. Phosphatidyl glycerol이 acyl phosphatidyl glycerol로 전환됨에 있어서 두 개의 다른 반응 기작을 밝혔다. 두 분자의 phosphatidyl glycerol이 한 분자의 acyl phosphatidyl glycerol 을 합성하는 즉 phosphatidyl glycerol이 acyl group의 공여체와 수용체로 각각 작용하는 반응과 다른 반응은 phosphatidyl glycer이 이 수용체로 phosphatidyl ethanolamine이 acyl group 공여체로 작용하는 기작이다. Acyl phosphatidyl glycerol 생합성에 있어서 최적 pH 는 7.0이며 최적 calcium ion 농도는 6.0mM 이었다. 이 실험 결과는 대장균의 phosphatidyl glycerol의 새로운 대사과정을 밝혔다. The present study has shown that phosphatidyl glycerol was converted to 3-sn-phosphatidyl-1'-(3'-acyl)-sn-glycerol (acyl phosphatidyl glycerol) by cell free extracts of Escherichia coli Band B/r. In this process, phosphatidyl glycerol was esterified directly rather than degraded to simpler compounds which are subsequently incorporated into acyl phosphatidyl glycerol. The enzyme, "acyl phosphatidyl glycerol synthetase", which catalyzes the conversion of phosphatidyl glycerol to acyl phosphatidyl glycerol was found to reside solely in the praticulate fraction of E. coli homogenates. Two separate reactions were identified where one molecule of acyl phosphatidyl glycerol was synthesized from two molecules of phosphatidyl glycerol, in which phosphatidyl glycerol acted both as an acyl acceptor and a donor. In the other reaction, phosphatidyl ethanolamine appears to act as an acyl donor to synthesize acyl phosphatidyl glycerol. The formation of acyl phosphatidyl glycerol has an optimum pH value at pH 7.0 and calcium concentration of 6.0 mM. These results show a new pathway for the turnover of phosphatidyl glycerol in Escherichia coli.

사람의 적혈구막의 Palmitoylcarnitine과 Palmitoyl-CoA : Carnitine Palmitine Palmitoyltransferase의 역할에 관한 연구

조기승,정진성,Cho, Key-Seung,Chung, Jin- Sung 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.3

An enzyme, palmitoyl-CoA: carnitine palmitoyltransferase (E.C. 2.3.1.21) was confirmed to catalyze reversively the formation and hydrolysis of $^{14}C$ -palmitoylcarnitine in human erythrocyte membrane. The optimum pH range of this enzyme was between from pH 7.2 to pH 7.4, and decreased rapidly outside of this pH range. In the several buffer systems tested, the highest enzyme activity was obtained with Tris-HCl buffer. As a cofactor, 5 ${\mu}moles$ of $Mg^{2+}$ ion significantly stimulated the enzyme activity in the experimental conditions. Palmitoylcarnitine formed from this enzyme system or synthesized from other chemical method has activated significantly the membrane bound enzymes, such as adenosine triphosphatase (ATPase) and Glucose-6-Phosphatase (G-6-Pase). 사람의 적혈구막에서 palmitoyl-CoA: carnitine palmitoyltransferase에 의해 $^{14}C$-palmitoylcarnitine이 가역적으로 생성 및 가수분해 됨을 확인하였다. 이 효소의 최적 pH 범위는 pH 7.2에서 pH 7.4 근방으로, 이 pH 범위 외에서는 급속한 효소활성도의 감소를 나타냈다. 몇가지의 완충용액을 시험하여 보았을때 Tris-HCI buffer가 가장 높은 효소활성도를 보였으며, 조요소로서 5 ${\mu}mole$의 $Mg^{2+}$이온이 동일 실험조건하에서 효소의 활성도를 굉장히 증가시켰다. 이 효소계에 의해서나 흑은 화학적인 합성에 의해 만들어진 palmitoyl. carnitine은 세포막에 부착되어 있는 효소인 ATPase나 G-6-Pase와 같은 효소를 상당히 활성화 시켰다.

사람 혈청의 Alkaline Phosphatase 에 대한 Ferrous 및 Ferric 이온과 여러 계면활성제의 영향

신현수,조기승 ( Hyun Soo Shin,Key Seung Cho ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

Alkaline phosphatase (E.C. 3.1.3.1) of human serum was shown to have a maximum activity with glycine buffer at pH 9. 8 and had a still high activity even with low and a little high temperature of 50℃. Although the enzyme was inhibited seriously in the presence of Cu^(++) and Fe^(++) ions, other cations tested showed little inhibition or activation effects. In comparison of Fe^(++) and Fe^(+++) ion`s effect on enzyme activity, Fe^(++) ion inhibited strongly, on the other hand, the enzyme was activated meaningfully by the addition of Fe^(+++) ion. This result suggested the possibility that ferrous and ferric ions in hemoglobin and methemoglobin, respectively, could control this enzyme activity in the blood stream. In the presence of EDTA, the enzyme activity was elevated significantly with both of Fe^(++) and Fe^(++) ions by the change of preincubation conditions, but EDTA itself had a property to inhibit completely the activity. Among several detergents tested, low concentration of palmitoylcarnitine, ginseng saponin and triton X-100 stimulated significantly the enzyme activity in the experimental conditions used, but the inhibition was shown in high concentration of above detergents and sodium deoxycholate.

사람 혈청의 Alkaline Phosphatase에 대한 Ferrous 및 Ferric 이온과 여러 계면활성제의 영향

신현수,조기승,Shin, Hyun-Soo,Cho, Key-Seung 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.2

사람 혈청의 alkaline phosphatase는 glycine buffer, pH 9.8에서 최고 활성도를 보였고, 저온이나 $50^{\circ}C$의 약간 높은 온도에서도 높은 활성도를 나타냈다. 여러 양이온에 대한 효과를 보면 $Cu^{++}$ 및 $Fe^{}$ 등에 의해서만 심한 저해 효과를 나타냈을 뿐, 시험한 다른 이온들은 약간의 저해 혹은 활성 효과를 나타냈다. $Fe^{++}$이온과 $Fe^{}$이온의 영향을 비교해 본 결과 $Fe^{++}$이온에 의해서는 심한 저해 효과를 보인 반면, $Fe^{}$이온은 현저한 활성 효과를 나타냈는데, 이 결과는 혈액내에서 hemoglobin과 methemoglobin의 ferrous 및 ferric 이온에 의해 효소의 활성도가 control될 수 있을 것이라는 점을 암시해 주고 있다. 한펀 EDTA 존재하에서의 $Fe^{++}$와 $Fe^{}$이온의 영향은 preincubation조건에 따라 두 이온이 모두 활성 효과를 보였는데, EDTA 단독 존재하에서는 철저한 저해 효과를 보였다. 여러 계면활성제의 영향을 보면, 저농도의 palmitoylcarnitine, 인삼 saponin 및 TX-100 등이 현저한 효소 활성 효과를 나타냈고, SDS는 약간의 활성 효과를 SDC는 저해 효과를 보여 주었다. Alkaline phosphatase (E.C. 3.1.3.1) of human serum was shown to have a maximum activity with glycine buffer at pH 9.8 and had a still high activity even with low and a little high temperature of $50^{\circ}C$. Although the enzyme was inhibited seriously in the presence of $Cu^{++}$ and $Fe^{++}$ ions, other cations tested showed little inhibition or activation effects. In comparison of $Fe^{++}$ and $Fe^{}$ ion's effect on enzyme activity, $Fe^{++}$ ion inhibited strongly, on the other hand, the enzyme was activated meaningfully by the addition of $Fe^{}$ ion. This result suggested the possibility that ferrous and ferric ions in hemoglobin and methemoglobin, respectively, could control this enzyme activity in the blood stream. In the presence of EDTA, the enzyme activity was elevated significantly with both of $Fe^{++}$ and $Fe^{}$ ions by the change of preincubation conditions, but EDTA itself had a property to inhibit completely the activity. Among several detergents tested, low concentration of palmitoylcarnitine, ginseng saponin and triton X-100 stimulated significantly the enzyme activity in the experimental conditions used, but the inhibition was shown in high concentration of above detergents and sodium deoxycholate.

인삼 사포닌의 생화학적 연구 ( ⅩⅧ ) 쥐 간 균질물에 의한 인 지질 생합성에 미치는 인삼 사포닌의 영향

박태경,주충노,조기승 ( Tae Kyung Park,Chung No Joo,Key Seung Cho ) 생화학분자생물학회 1981 BMB Reports Vol.14 No.4

It has been attempted in this study to observe the effect of ginseng saponin on phospholipid biosynthesis by rat liver homogenate using (U-^(14)C)-glycerol, (U-^(14)C) -snglycerol-3-phosphate, and (1-^(14)C)-palmityl CoA as tracer in vitro and the following results were obtained. 1. Biosynthesis of phosphatidyl etanolamine using (U-^(14)C)-glycerol as tracer was increased as much as 10 times when the concentration of ginseng saponin in the reaction mixture was 10^(-4)∼10^(-3). 2. In case of experiment using (U-^(14)C)-sn-glycerol-3-phoshate as tracer, biosoynthesis of total lipids was also stimulated at concentration of 10^(-4)∼10^(-3)% of ginseng saponin. Furthermore, biosynthesis of phosphatidyl ethanolamine and phophatidyl choline was increased in the reaction mixture containing 10^(-4)% ginseng saponin. 3. Using liver homogenate and (1-^(41)C)-palmityl CoA only, it was observed that the ginseng saponin (10^(-4)∼10^(-3)%) stimulated the biosynthesis of phosphatidyl choline and phosphatidyl ethanolamine. From the above experimental results, it seemed that adequate amounts of ginseng saponin might stimulate the biosynthesis of phospholipids significantly.

적혈구 막에 관한 연구(I) Palmitoyl Carnitine에 의한 소 적혈구 막의 용해

이강순,장정순,조기승,이강석,Rhee, Kang-Soon,Chang, Chung-Soon,Cho, Key-Seung,Lee, Kang-Suk 생화학분자생물학회 1975 한국생화학회지 Vol.8 No.1

소의 적혈구막을 palmitoyl carnitine으로 처리하였을 때 단백질, 인지질 및 cholesterol이 동시에 완전히 용해되었고 palmitoyl carnitine에의 한 지질의 유리 및 micelle의 형성이 없는 것으로 보아 적혈구막은 lipoprotein 형태로 용해됨을 알았다. 소 적혈구막의 용해는 palmitoyl carnitine이 막의 lipoprotein과의 친수성 결합과 소수성 결합을 하므로써 용해되며 용해도는 소수성 결합 정도에의 존한다고 본다. Palmitoyl carnitine에의 한 혈구막 용해에 있어서 ATPase($Na^+$, $K^+$ dependent)와 glucose-6-phosphatase의 활성도는 저해 받지 않았으며 오히려 palmitoyl carnitine의 농도에 따라 각각 28-43%, 56-110%가 증가하였다. 용해된 막단백질에 있어 pH에 따른 용해도의 상태는 pH 5.0에서 92%의 단백질이 침전되었다. 용해된 막단백질을 Tris-glycine buffer, pH 8.3로 polyacrylamide gel electrophoresis를 행하였을 때 분자량이 450,000-70,000인 8개의 band로 분리되었다. The mechanism of solubilization of the ox-erythrocyte membrane was investigated with palmitoyl carnitine. When the membrane was treated with palmitoyl carnitine, the membrane protein, phospholipids and cholesterol were completely solubilized. It was confirmed that lipids were not liberated from membrane lipoprotein and not form micelles with palmitoyl carnitine. As a consequence, membrane was solubilized as a form of lipoprotein. In the present study, the results indicated that the erythrocyte membrane was solubilized by involvement of ionic and hydrophobic interaction of membrane lipoprotein with palmitoyl carnitine. The solubility of erythrocyte membrane depended upon the degree of the hydrophobic binding with palmitoyl carnitine. In the complete solubilized membrane, the activities of ATPase($Na^+$, $K^+$-dependent) and glucose-6-phosphatase were not affected or rather slightly increased of 28% and 56% as compared with intact membrane, respectively. The effect of pH on solubilized membrane protein showed that 92% of protein was precipitated at pH 5.0. In polyacrylamide gel electrophoresis with Tris-glycine buffer, pH 8.3, solubilized membrane protein was separated into 8 bands with molecular weight ranged from 450, 000 to 70, 000.

생쥐뇌 세포질 분획의 Acid 및 Alkaline Phosphatase 의 특성 비교 연구

강창균,이영식,조기승 ( Chang Gyun Kang,Young Seek Lee,Key Seung Cho ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.3

Acid and alkaline phosphatases from mouse brain cytosol fraction were separated and partially purified with 91 and 15 folds, respectively, by DEAE-cellulose column chromatography and Sephadex G-150 gel chromatography. Acid phosphatase (ACPase, EC 3.1.3.2) showed substrate specificity only to p-nitrophenylphosphate among the several substrates tried, such as, adenosine-3`-monophosphate (3`-AMP), adenosine-5`-monophosphate (5`-AMP), adenosine-5`-triphosphate (5`-ATP), glucose-6-phosphate (G-6-P), inositol-1`-monophosphate (IP), inositol-1,4,5-triphosphate (IP₃ and p-nitrophenylphosphate (PNPP). On the other hand, alkaline phosphatase (ALPase, EC 3.1.3.1) showed to active to PNPP and IP substrates among them. ACPase and ALPase showed the maximum enzyme activities at pH 5.2 with 50 mM acetate buffer and at pH 7.8 with 50 mM Hepes buffer in the presence of Mg^(2+), respectively. In the effect of Na^+ and K^+ on both ACPase and ALPase, these ions had no effect at all on ACPase, but 50 mM Na^+ and 100 mM K^+ showed maximum activation on ALPase by 3.4 times. Divalent cations,Ca^(2+) and Mg^(2+) showed no effect at all on ACPase, but 0.2 mM Mg^(2+) stimulated ALPase significantly about 5 times. On the other hand, Ca^(2+) represented competitive inhibition to Mg^(2+) effect on ALPase. Ouabain had no effect at all, but L-phenylalanine showed a minor stimulation on ALPase.

생쥐 간의 Triacylglycerol 생합성에 있어서 Palmitoylcarnitine의 지방산 공여체로서의 역할

이윤경,정진성,조기승,Lee, Youn-Kyung,Chung, Jin-Sung,Cho, Key-Seung 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3

$^{14}C$-Palmitoylcarnitine을 생쥐 간 균질물과 반응시킨 결과 1시간 후 33%, 3시간 후에 51%, 6시간 후에 최고 61%의 가수분해를 나타냄으로서, 동물체의 간이 acylcarnitine의 중요한 대사부위 임을 나타내 주었다. $^{14}C$-Palmitoylcarnitine ($^{14}C$-pal. carn)에 의한 $^{14}C$-triacylglycerol ($^{14}C$-TG)의 생합성은 0.1 M tris-HCl buffer, pH 7.4 및 반응온도 $20^{\circ}C$에서 각각 최고의 합성율을 나타냈고, ATP, coenzyme A (CoASH), $Ca^{2+}$ 및 $Mg^{2+}$의 cofactor 등은 triacylglycerol 생 합성계에 전혀 영향을 끼치지 않았으며, 이들은 phospholipid 생성계를 활성화시키는 것으로 나타났다. 동일 조건하에서 (U-$^{14}C$)-palmitic acid에 의한 중성지 질의 생합성을 비교하여 본 결과, 반응온도는 $20^{\circ}C$가 적정온도였고, 이 반응계는 ATP, CoASH 및 $Mg^{2+}$의 cofactor를 필요로 했는데 반응시간에 따라 차이를 보여주었다. 즉, 30분 반응에서는 cofactor 존재하에서 $^{14}C$-TG 생성이 증가를 보였으나, 3시간 반응에서는 cofactor가 존재하지 않은 대조구보다 $^{14}C$-phospholipids 생성이 증가함을 나타냈다. 간 균질물의 여러 분획에 있어서 $^{14}C$-pal. carn에 의한 $^{14}C$-TG의 생성을 비교해 본 결과, microsomal cytosol 분획에서 가장 높은 triacylglycerol와 diacylglycerol의 생성율을 보였고, 다음이 mitochondria 분획, nuclei와 cell debris 분획 순이었다. 이상의 결과로 보아 palmitoylcarnitine은 microsome에서 cofactor의 도움 없이 직접 acyl group을 이전시켜 중성지질을 생성하는 것을 확인할 수 있었다. It was confirmed that the important metabolic site of acylcarnitine was liver in mamals from the results of its hydrolysis, in which percent of hydrolysis was 33%, 51 %, and 61% from incubation for 1 h, 3 h, and 6 h, respectively. In the triacylglycerol biosynthesis from (U-$^{14}C$)-palmitolyl-DL-carnitine, the optimum conditions were obtained in 0.1 M tris-HCl buffer, pH 7.4 and $20^{\circ}C$ of incubation temperature. The effect of cofactors, such as $Ca^{2+}$, $Mg^{2+}$, ATP, and coenzyme A reduced the formation of triacylglycerol, rather increased the formation of phospholipid. This result showed that triacylglycerol synthesis from palmitolycarnitine did not need any cofactors. The comparison of the triacylglycerol biosynthesis from (U-$^{14}C$)-palmitic acid with that of (U-$^{14}C$)-palmitoyl-DL-carnitine showed the same optimum conditons in pH and incubation temperature. But in cofactors effect, tracylglycerol synthesis from $^{14}C$-palmitic acid was stimulated in the presence of ATP, CoASH, and $Mg^{2+}$ in 30 min incubation when it was compared with or without of cofactors. When the incubation period was prolonged to 3 h, triacylglycerol synthesis was reduced and, on the other hand, phospholipid synthesis was increased significantly more than 6 times. With the results from the different cell fractions, the highest triacylglycerol biosynthesis was prepresented with microsomal cytosol fraction and next was in order of mitochondrial fraction and nuclei cell debris fraction. As conclusion, firstly, it could be postulated that the direct mobilization of acyl group from palmitoylcarnitine to monoacylglycerol and diacylglycerol resulted triacylglycerol synthesis. Secondly, free fatty acid from hydrolysis of palmitoylcarnitine might from acyl CoA in the presence of ATP and CoASH, and continued the sequencial reactions with diacylglycerol and with Iysophospholipid to form the triacylglycerol and phospholipid, respectively.