해녀콩 ( Canavalia lineata ) 잎에서 Arginase 활성의 세포내 분포

유경희(Gyung Hee Yu),권영명(Young Myung Kwon) 한국식물학회 1990 Journal of Plant Biology Vol.33 No.1

Subcellular distribution of arginase activity was measured in leaves of Canavalia lineata. Both mitochondrial and cytosolic fraction were found to contain the arginase activity. It was noticible that cytosolic fraction contained a substantial amount of arginase activity. Different mobility of arginase from two fractions was showed on DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. Also different pI value was showed 6.3 in cytosolic and 6.7, 7.1 in mitochondrial fraction on IEF gel electrophoresis. However, canavanine-dependent-activity (CDA) of arginase in these two fractions were not different. These results indicate that heterogenity of arginase occurs in leaves of C. lineata.

Alteration of Arginase Activity in Leaf Protoplasts of Canavalia lineata

유경희,권영명,Yu, Gyung-Hee,Kwon, Young-Myung 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.3

해녀콩(Canavalia lineata L. (DC))의 잎절편에서 incubation 용액의 pH가 6에서 8로 높아짐에 따라 arginase 활성이 현저하게 증가하였다. Arginase 활성에 따른 arginine과 canavanine의 함량변화를 초엽에서 원형질체를 분리하여 조사하였다. 전유리아미노산 함량 중 canavanine과 arginine은 각각 30%와 5%를 차지하였고, 세포에 존재하는 이들 두 아미노산의 60% 이상이 액포에 존재하였다. 원형질체를 10 mmolar [U-$^{14}C$]arginine이 첨가된 용액에서 1시간 동안 incubation하면 pH 6의 용액보다 pH 8의 용액에서 [$^{14}C$]ornithine이 더 많은 양이 축적되었고, 이때 세포내 arginine의 함량은 감소함을 보였다. Arginine을 첨가하지 않고 1시간 동안 incubation한 원형질체에서는 canavanine과 arginine의 함량은 pH 8의 용액에서는 50% 감소하였지만, pH 6의 용액에서는 전혀 함량감소가 일어나지 않았다. 본 실험결과에서 원형질체를 알칼리 pH용액에서 incubation하면 arginase 활성의 현저한 증가와 더불어 canavanine과 arginine의 pool이 감소함을 알 수 있었다. Arginase activity in the leaf discs of Canavalia lineata L. (DC) markedly increased in response to increasing the medium pH from 6 to 8. The changes in levels of arginine and canavanine in relation to arginase activity were investigated in protoplasts isolated from primary leaves. Canavanine and arginine accounted for about 30% and 5% of total free amino acids, respectively, and more than 60% of cellular canavanine and arginine was located in the vacuole. The incubation of protoplasts for 1 h with 10 mmolar [U-$^{14}C$]arginine at pH 8 resulted in a greater accumulation of [$^{14}C$]ornithine than that at pH 6 accompanied by a lower cellular arginine level. In the protoplasts incubated for 1 h without exogenous arginine, levels of canavanine and arginine decreased by 50% at pH 8 while their levels did not decrease at pH 6. These results suggest that incubation of protoplasts at an akaline pH gives rise to not only a significant increase in arginase activity but also depletion of pools of canavanine and arginine.

녹화중 보리유식물에서 Phosphorylcholine 대사의 변화

유경희(Gyung Hee Yu),권영명(Young Myung Kwon) 한국식물학회 1988 Journal of Plant Biology Vol.31 No.4

We investigated the activities of choline kinase, CTP: phosphorylcholine cytidyltransferase, and phosphatase during the greening of etiolated barley seedlings. Activities of choline kinase in leaves increasd until 6 hours after illumination and decreased considerably after 6 hours, while activities of CTP: phosphorylcholine cytidyltransferase increased after illumination. On the contrary, changes of these two enzymatic activities showed reverse pattern in roots. The activities of phosphatase which hydrolyze phosphorylcholine decreased in leaves but changed little in roots during greening. The concentration of phosphorylcholine increased in xylem exudate and in roots during greening, while decreased in leaves. These results suggested that more phosphorylcholine arrive in leaves from roots as greening of etiolated barley seedlings.

해녀콩잎 원형질에서 pH 변화에 의한 Arginase 의 활성변화

유경희,권영명 ( Gyung Hee Yu,Young Myung Kwon ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.3

Arginase activity in the leaf discs of Canavalia lineata L. (DC) markedly increased in response to increasing the medium pH from 6 to 8. The changes in levels of arginine and canavanine in relation to arginase activity were investigated in protoplasts isolated from primary leaves. Canavanine and arginine accounted for about 30% and 5% of total free amino acids, respectively, and more than 60% of cellular canavanine and arginine was located in the vacuole. The incubation of protoplasts for 1 h with 10 mmolar [U-^(14)C]arginine at pH 8 resulted in a greater accumulation of [^(l4)C]ornithine than that at pH 6 accompanied by a lower cellular arginine level. In the protoplasts incubated for 1 h without exogenous arginine, levels of canavanine and arginine decreased by 50% at pH 8 while their levels did not decrease at pH 6. These results suggest that incubation of protoplasts at an akaline pH gives rise to not only a significant increase in arginase activity but also depletion of pools of canavanine and arginine.

Phosphorylcholine 이 보리 엽록체의 광인산화활성에 미치는 저해효과에 관하여

유경희(Gyung Hee Yu),이진범(Chin Bum Lee),권영명(Young Myung Kwon) 한국식물학회 1986 Journal of Plant Biology Vol.29 No.3

The onset of photophosphorylation at the various stages of greening showed different patterns with varying concentrations of Pi. With further greening. ATP formation occurred at the lower concentration of Pi (48 hrs; 0.05 mM). At early stages of greening, more Pi was required for photophosphorylation (6 hrs; 5.0 mM). The addition of cell-free extracts of etiolated barley seedlings resulted in the competitive inhibition of photophophorylation. The apparent inhibition by cell-free extracts was gradually decreased during greening of etiolated barley seedlings. We found that the inhibitors of photophosphorylation in cell-free extracts were some organic phosphates and most of them was P-choline. P-choline inhibited photophosphorylation competitively with Pi and its content was decreased considerably in greening. It is likely that P-choline partly delay the photophosphorylation in early stages of greening.

Phosphorylcholine , Phosphorylethanlamine 및 chlorocholine 이 보리 엽록체의 광인산화활성에 미치는 저해효과의 비교

유경희,이진범,권영명 ( Gyung Hee Yu,Chin Bum Lee,Young Myung Kwon ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3

The activity of photophosphorylation of isolated chloroplasts from barley leaves was apparently inhibited by phosphorylcholine; phosphorylcholine drastically reduced ATP synthesis and irreversible proton consumption accompanying ATP synthesis. But, phosphorylethanolamine and chlorocholine showed little inhibitory effects on photophosphorylation. Phosphorylcholine did not affect electron transport activities and increased buffer capacity of barley chloroplast suspensions, as phosphorylethanolamine and chlorocholine did. The leakage of thylakoid membranes to proton was induced larger amount by phosphorylethanolamine and chlorocholine rather than by phosphorylcholine. It appeared that this leakage could not cause a marked inhibition of photophosphorylation. These results support an assumption that phosphorylcholine has inhibitory effects on coupling factor and thylakoid membranes while the other two compounds act on thylakoid membranes.

Phosphorylcholine, Phosphorylethanolamine 및 Chlorocholine이 보리 엽록체의 광인산화활성에 미치는 저해효과의 비교

유경희,이진범,권영명,Yu, Gyung-Hee,Lee, Chin-Bum,Kwon, Young-Myung 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3

Phosphorylcholine은 보리에서 분리한 엽록체의 광인산화활성에 현저한 저해효과를 보였다. 즉 광인산화반응시 ATP 생성이 억제됨과 동시에 ATP생성으로 인하여 비가역적으로 소비되는 proton의 양도 감소하는 것을 볼 수 있었다. 그러나 광안산화활성에 대한 phosphoryl ethanolamine, chlorocholine의 저해효과는 낮은 것으로 나타났다. Phosphorylcholine, phosphorylethanolamine, chlorocholine 모두는 전자전달 반응계에 전혀 영향을 미치지 않았으며 엽록체 반응액의 buffer capacity를 증가시키기도 하였다. Phosphorylcholine은 엽록체의 틸라코이드막의 proton leakage를 약간 촉진하였지만 phosphorylethanolamine, chlorocholine의 경우와 비교하면 상대적으로 낮은 수준이었다. 따라서 이러한 proton leakage효과가 광인산화활성을 뚜렷하게 억제시킬 정도는 아님을 알 수 있었다. 이상의 결과에서 phosphorylethanolamine, chlorocholine은 틸라코이드막의 수준에서 비특이적인 저해효과를 미친 반면에 phosphorylcholine은 틸라코이드막뿐만 아니라 특히 coupling factor에 저해적으로 작용하여 광인산화활성을 억제하는 것으로 추정할 수 있었다. The activity of photophosphorylation of isolated chloroplasts from barley leaves was apparently inhibited by phosphorylcholine; phosphorylcholine drastically reduced ATP synthesis and irreversible proton consumption accompanying ATP synthesis. But, phosphorylethanolamine and chlorocholine showed little inhibitory effects on photophosphorylation. Phosphorylcholine did not affect electron transport activities and increased buffer capacity of barley chloroplast suspensions, as phosphorylethanolamine and chlorocholine did. The leakage of thylakoid membranes to proton was induced larger amount by phosphoryl ethanolamine and chlorocholine rather than by phosphorylcholine. It appeared that this leakage could not cause a marked inhibition of photophosphorylation. These results support an assumption that phosphorylcholine has inhibitory effects on coupling factor and thylakoid membranes while the other two compounds act on thylakoid membranes.

해녀콩 ( Canavalia lineata ) 자엽 Arginase 의 정제와 효소학적 특성

유경희,전방욱,홍영남,권영명 ( Gyung Hee Yu,Bang Ook Jun,Young Nam Hong,Young Myung Kwon ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) from cotyledones of Canavalia lineata was purified and characterized. The purification steps involved streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography, canavanine-Sepharose 4B affinity chromatography, and Sephadex G200 gel filtration chromatography. Arginase was purified 217-fold with 4% recovery. These column chromatography revealed that ADA (arginine-dependent activity) and CDA (canavanine-dependent activity) eluted the same peak. The pH optimum was 9 for ADA and 8 / or 8.5 for CDA. Kinetic analyses of purified arginase for arginine revealed an apparent K_m of 30 mM at pH 9 and 82 mM at pH 8. Comparable determinations with canavanine revealed an apparent K_m of 62 mM at pH 9 and 43 mM at pH 8. It is suggested that the affinity for canavanine with this enzyme at physiological pH be as high as that for arginine. 1 mM Mn^(2+) was required for the optimal enzyme activity and maximum activity was exerted at 40℃. The molecular weight was estimated as 180,000 by Sephadex G200 gel filtration chromatography and subunit molecular weight was obtained as 44,000 by SDS-PAGE.

해녀콩(Canavalia lineata) 자엽 Arginase 의 정제와 효소학적 특성

유경희,전방욱,홍영남,권영명,Yu, Gyung-Hee,Jun, Bang-Ook,Hong, Young-Nam,Kwon, Young-Myung 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

해녀콩 (Canavalia lineata ) 자엽의 arginase (L-arginine amidino hydrolase, EC3.5.3.1)를 정제하여서 그 특성을 조사하였다. 이 효소는 황산스트렙토마이신 침전, $AmSO_4$ 분획, DEAE-Sephacel, canavanine-Sepharose 4B affinity gel 및 Sephadex G-200 gel 크로마토그래피를 한 결과 4%의 회수율로 fold는 217배 증가되었다. 이상 3 종류의 컬럼크로마토그래피에서 ADA (arginine-dependent activity)와 CDA (canavanine-dependentactivity)의 활성은 항상 같은 fraction에서 peak를 보였다. 그리고 최적 pH는 ADA의 경우9.0이었고 CDA의 경우 8.0과 8.5이었다. 또한 arginine에 대한 $K_m$은 pH 9.0에서 30 mM, pH 8.0에서 82 mM이였다. 이에 비하여 canavanine에 대한 효소의 $K_m$은 pH 9.0에서 62 mM, pH 8.0에서 43 mM이었다. 이러한 결과에서 생리적인 pH에서는 canavanme에 대한 효소의 친화성이 arginine에 대한 친화성 만큼 높은 것으로 추정할 수 있었다. 1 mM $Mn^{2+}$일 때 효소의 활성이 최대로 나타났으며 최적온도는 $40^{\circ}C$이었다. Sephadex G-200 gel 컬럼에서 arginase의 분자량은 180,000이었으며, SDS-PAGE에서 얻은 subunit의 분자량은 44,000임을 알 수 있었다. Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) from cotyledones of Canavalia lineata was purified and characterized. The purification steps involved streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography, canavanine-Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Arginase was purified 217-fold with 4% recovery. These column chromatography revealed that ADA (arginine-dependent activity) and CDA (canavanine-dependent activity) eluted the same peak. The pH optimum was 9 for ADA and 8 / or 8.5 for CDA. Kinetic analyses of purified arginase for arginine revealed an apparent $K_m$, of 30 mM at pH 9 and 82 mM at pH 8. Comparable determinations with canavanine revealed an apparent $K_m$, of 62 mM at pH 9 and 43 mM at pH 8. It is suggested that the affinity for canavanine with this enzyme at physiological pH be as high as that for arginine. 1 mM $Mn^{2+}$ was required for the optimal enzyme activity and maximum activity was exerted at $40^{\circ}C$. The molecular weight was estimated as 180,000 by Sephadex G-200 gel filtration chromatography and subunit molecular weight was obtained as 44,000 by SDS-PAGE.