La3+에 의한 토마토 뿌리조직 마이크로솜 H+-ATPase 활성저해

조광현 ( Kwang Hyun Cho ),김영기 ( Young Kee Kim ) 한국응용생명화학회 2003 Applied Biological Chemistry (Appl Biol Chem) Vol.46 No.2

In order to find a chemical agent which is able to modulate the activity of H^+-ATPase, microsomal preparation was obtained from the root tissue of tomato plant and the effect of La^3+ was measured. The activities of plasma and vacuolar membrane H^+-ATPase were analyzed by the inhibited activities using their specific inhibitors, vanadate and NO_3^-, respectively. La^3+ inhibited microsomal ATPases in a dose-dependent manner and the inhibitory effect of La^3+ was suppressed by both vanadate and NO_3^-, implying that La^3+ inhibits both plasma and vacuolar membrane H^+-ATPase. The Ki values of La^3+ which inhibit 50% of the activities of plasma and vacuolar membrane H^+-ATPase were 57 and 78μM, respectively. The H^+-ATPase of the leaky microsomes made by the treatment of Triton X-100 were also inhibited by La^3+, suggesting that La^3+ directly inhibits both enzymes. Meanwhile, the inhibitory effect of La^3+ was decreased by increasing the concentration of ATP. The effect of ATP was also concentration-dependent and 7 mM ATP completely removed the inhibitory effect of La^3+. These results imply that La^3+ inhibits both plasma and vacuolar membrane H^+-ATPase by decreasing the binding affinity of ATP and La^3+ can be used to control the activity of root H^+-ATPase.

$La^{3+}$에 의한 토마토 뿌리조직 마이크로솜 $H^+-ATPase$ 활성저해

조광현,김영기,Cho, Kwang-Hyun,Kim, Young-Kee 한국응용생명화학회 2003 한국농화학회지 Vol.46 No.2

$H^+-ATPase$ 활성을 조절할 수 있는 물질을 찾기 위하여 토마토 뿌리조직으로부터 마이크로솜을 분리하고 $La^{3+}$의 효과를 조사하였다. 원형질막 및 액포막에 위치하는 $H^+-ATPase$의 활성은 각각의 선택적 저해제인 vanadate와 $NO_3-$의 처리시 감소하여, $La^{3+}$이 원형질막 및 액포막 $H^+-ATPase$ 활성을 모두 저해함을 확인하였다. 원형질막과 액포막 $H^+-ATPase$ 활성을 50% 저해하는 $La^{3+}$ 농도인 Ki 값은 각각 57, $78\;{\mu}M$이었다. $La^{3+}$에 의한 저해효과는 Triton X-100을 처리한 leaky 마이크로솜에서도 얻어져, $La^{3+}$이 이온채널의 존재와 관계없이 $H^+-ATPase$의 활성을 직접적으로 저해함을 확인하였다. 한편, Lak의 활성저해 효과는 ATP 농도 증가로 감소하였고, ATP의 효과는 농도 의존적으로 나타났으며, 7 mM ATP 의해 $La^{3+}$에 의한 $H^+-ATPase$ 활성 저해가 완전히 억제되었다. 이러한 결과로부터 $La^{3+}$은 원형질막과 액포막의 $H^+-ATPase$들에 결합하여 ATP 결합친화력을 감소시킴으로써 활성을 저해하며, 뿌리조직 $H^+-ATPase$의 활성조절제로 이용이 가능함을 확인하였다. In order to find a chemical agent which is able to modulate the activity of $H^+-ATPase$, microsomal preparation was obtained from the root tissue of tomato plant and the effect of $La^{3+}$ was measured. The activities of plasma and vacuolar membrane $H^+-ATPase$ were analyzed by the inhibited activities using their specific inhibitors, vanadate and $NO_3-$, respectively. $La^{3+}$ inhibited microsomal ATPases in a dose-dependent manner and the inhibitory effect of $La^{3+}$ was suppressed by both vanadate and $NO_3-$, implying that $La^{3+}$ inhibits both plasma and vacuolar membrane $H^+-ATPase$. The Ki. values of $La^{3+}$which inhibit 50% of the activities of plasma and vacuolar membrane $H^+-ATPase$ were 57 and $78\;{\mu}M$, respectively. The $H^+-ATPase$ of the leaky microsomes made by the treatment of Triton X-100 were also inhibited by $La^{3+}$, suggesting that $La^{3+}$ directly inhibits both enzymes. Meanwhile, the inhibitory effect of $La^{3+}$ was decreased by increasing the concentration of ATP, The effect of ATP was also concentration-dependent and 7 mM ATP completely removed the inhibitory effect of $La^{3+}$. These results imply that $La^{3+}$ inhibits both plasma and vacuolar membrane $H^+-ATPases$ by decreasing the binding affinity of ATP and $La^{3+}$ can be used to control the activity or root $H^+-ATPases$.

생화학,분자생물학 : 토마토 뿌리조직 H(+)-ATPase 활성에 미치는 Thapsigargin의 저해효과

조광현 ( Kwang Hyun Cho ),김영기 ( Young Kee Kim ) 한국응용생명화학회 2005 Applied Biological Chemistry (Appl Biol Chem) Vol.48 No.3

생화학,분자 생물학 : Zn(2+)에 의한 Tolaasin의 용혈활성 저해효과

조광현 ( Kwang Hyun Cho ),김성태 ( Sung Tae Kim ),김영기 ( Young Kee Kim ) 한국응용생명화학회 2006 Applied Biological Chemistry (Appl Biol Chem) Vol.49 No.4

토마토 뿌리조작에서 분리한 마이크로솜의 Ca2+ 흡수 특성

조광현(Kwang Hyun Cho),김영기(Young Kee Kim) 한국응용생명화학회 1999 Applied Biological Chemistry (Appl Biol Chem) Vol.42 No.2

In order to characterize the property of Ca^(2+) transport in plant cell, microsomes were prepared from the roots of tomato and microsomal ^(45)Ca^(2+) uptake was measured. When 1 mM vanadate, a selective inhibitor of P-type ATPases, 50 mM NO₃^-, a specific inhibitor of vacuolar H^+-ATPase, and both of these inhibitors were treated, the microsomal ^(45)Ca^(2+) uptakes were inhibited by 20, 33 and 47%, respectively. The inhibitory effects of these two inhibitors were investigated by using a protonophore, gramicidin. When the chemical gradient of H^+ was relieved by gramicidin, the uptake was decreased by 30%, implying the presence of Ca^(2+)/H^+ antiporter in the microsomal membrane. In the ^(45)Ca^(2+) uptake experiment, the effect of gramicidin was independent of vanadate-induced inhibition. However, when the activity of vacuolar H^+-ATPase was inhibited by NO₃^-, the effect of gramicidin was severely decreased. Meanwhile, thapsigargin, a specific antagonist of ER/SR-type Ca^(2+)-ATPase, inhibited the microsomal ^(45)Ca^(2+) uptake and the maximum inhibitory effect was obtained at 10 μM. The effect of thapsigargin was blocked by NO₃^- and gramicidin, but not by vanadate. These results imply that vanadate directly inhibits the activity of Ca^(2+)-ATPase; however, NO₃^- and thapsigargin block the activity of Ca^(2+)/H^+ antiporter by inhibiting the vacuolar H^+-ATPase. In conclusion, the microsomal ^(45)Ca^(2+) uptakes are mediated by two major enzymes, Ca^(2+)-ATPase and Ca^(2+)/H^+ antiporter in tomato root tissue.

Quinacrine 형광을 이용한 토마토 뿌리조직 마이크로솜의 수소이온이동 활성측정

신대섭,조광현,김영기,Shin, Dae-Seop,Cho, Kwang-Hyun,Kim, Young-Kee 한국응용생명화학회 2002 한국농화학회지 Vol.45 No.2

Quinacrine은 수소이온 농도변화에 민감한 형광 probe로서 양성자와 결합하지 않은 형광형이나, 양성자와 결합한 비형광형으로 존재한다. 따라서, quinacrine은 $H^+-ATPase$에 의한 수소이온이동 활성 측정에 이용된다. 본 연구에서는 토마조 뿌리조직에서 분리한 마이크로솜에서 quinacrine의 형광성을 이용한 $H^+-ATPase$ 활성측정의 최적 조건을 조사하였다. Quinacrine의 형광변화는 반응용액 중의 단백질 함량이 $0.43{\mu}g/{\mu}l$에서25-26% 감소하여 10%의 quinacrine 형광을 감소시키는 데 약 100nmo1/min의 $H^+-ATPase$ 활성이 필요함을 알 수 있었다. Quinacrine의 최대 형광변화는 pH 7.0-7.2 범위와 $2mM\;Mg^{2+}$ 조건에서 일어났다. 이것은 기존에 보고한 $H^+-ATPase$의 특성과 일치하여, quinacrine의 형광변화가 $H^+-ATPase$의 활성을 잘 반영하고 있음을 보인다. 원형질막 및 액포막 $H^+-ATPase$들의 선택적 저해제인 vanadate와 $NO_3-$는 각각의 효소에 의한 수소이온이동 활성을 저해하는데 성공적임을 확인하였다. 이상의 결과로 quinacrine이 토마토 뿌리조직에서 분리한 마이크로솜의 수소이온이동 활성측정에 유용하게 이용될 수 있음을 확인하였다. Quinacrine, a pH-sensitive fluorescence probe, which exists either as an unprotonated fluorescence form or a protonated noufluorescence form, can be used to measure the proton transport activity of $H^+-ATPase$. Quinacrine was used to determine the optimal conditions for measuring the activity of microsomal $H^+-ATPase$ prepared from the roots of tomato plants. The amount of quinacrine fluorescence quenching obtained at $0.43{\mu}g/{\mu}l$ of microsomal protein concentration was 25-26%, which shows that the enzyme activity of 100 nmol/min decreases 10% of quinacrine fluorescence. Maximal fluorescence quenching was obtained at pH 7.0-7.2 and 2 mM $Mg^{2+}$ Because the activity of microsomal $H^+-ATPase$ is also maximal at these conditions, the quinacrine fluorescence well represents the activity of $H^+-ATPase$. Vanadate and $NO_3-$, specific inhibitors of plasma and vacuolar $H^+-ATPases$, respectively, were successfully applied to inhibit the quinacrine fluorescence quenching mediated by the corresponding $H^+-ATPases$. These results imply that quinacrine is a useful tool for measuring the proton transport activities of microsomes obtained from the root tissue of tomato plants.

토마토 뿌리조직에서 분리한 마이크로솜 이온펌프의 특성

조광현,사공정,김영기 ( Kwang Hyun Cho,Jung Sakong,Young Kee Kim ) 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.41 No.2

Microsomes of tomato roots were prepared and the activities of microsomal ATPases were measured in order to understand the molecular mechanisms of various ion transports. The activities of plasma membrane H^+-ATPase and vacuolar H^+-ATPase were evaluated to ∼30% and ∼38% of total microsomal ATPase activity by using their specific inhibitor, vanadate and nitrate (NO₃^-), respectively. The inhibitory effects of vanadate and NO₃^- were additive and the simultaneous additions of these two inhibitors decreased the total activity up to 50∼70%. The microsomal ATPase activity was regulated key pH and the maximal activity was obtained at pH 7.4. The activity of microsomal ATPase was increased by K^+ up to ∼30% at the concentration of K^+ above 10 mM. However, the K^+- induced increase in the activity was Completely inhibited by the simultaneous addition of Na^+. To identify the ATPase activity regulated by K^+, the effects of specific inhibitors were measured. Vanadate and NO₃^- inhibited total ATPase activity by 27% and 32% in the absence, of K^+ and by 27% and 40% in the presence of 120 mM K^+, respectively. These results suggest that K^+ increases the activity of NO₃^--sensitive vacuolar H^+-ATPase but not that of vanadate-sensitive plasma membrane H^+-ATPase since vanadate has no effect on K^+-induced increase in ATPase activity. The microsomal ATPase activity was also decreased by increasing Ca^(2+) concentration. Interestingly, NO₃^- blocked the Ca^(2+)-induced inhibition of microsomal ATPase activity; however, vanadate had no effect. These results imply that vacuolar H^+-ATPase is activated by K^+ and inhibited by Ca^(2+).

콩 뿌리조직에서의 Ca2+에 의한 마이크로솜 이온펌프 활성저해

조광현,조경수,이은형,김영기 ( Kwang Hyun Cho,Kyoung Soo Cho,Eun Hyoung Lee,Young Kee Kim ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.3

In order to investigate the mechanisms of epithelial ion transports, microsomes of soybean roots were prepared and the activity of microsomal ATPases was measured by an enzyme-coupled assay. The effects of various ions were evaluated on the total activity of microsomal ATPases and the average activity was 190 nmol/min/㎎ protein in the control solution containing 10 mM Na^+ and 120 mM K^+. The activities were increased to 150% and decreased to 63% of the control activity in the solution containing 130 mM K^+ without Na^+ and in the solution containing 130 mM Na^+ without K^+, respectively. In general, the activity of microsomal ATPase was increased by K^+ in a concentration-dependent manner. The activity was also increased at lower pH and relatively higher activities were observed in the pH range of 6∼7. However, the activity was decreased at weak alkaline pH and ∼80% of the activity was inhibited at pH 9. Since intracellular Ca^(2+) has been known to control the activity of various enzymes, we have investigated the effects of intra- and extramicrosomal Ca^(2+) on the activity of microsomal ATPases. The maximal activity was obtained at the extramicrosomal Ca^(2+) concentrations below 1 nM. The activity was gradually decreased by increasing Ca^(2+) concentration and 50% inhibition was observed at ∼500 μM Ca^(2+). The increase in luminal Ca^(2+) concentration also inhibited the activity of microsomal ATPase. When the influx of external Ca^(2+) was induced by Ca^(2+) ionophore A23187 treatment, the activity was decreased by 30%; however, it was recovered by EGTA-induced chelation of Ca^(2+). These results suggest that the presence of Ca^(2+) regulation sites on both cytoplasmic and luminal sides of microsomal ATPases.

콩 뿌리조직에서의 $Ca^{2+}$에 의한 마이크로솜 이온펌프 활성저해

조광현,조경수,이은형,김영기,Cho, Kwang-Hyun,Cho, Kyoung-Soo,Lee, Eun-Hyoung,Kim, Young-Kee 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.43 No.2

콩 뿌리조직의 이온 흡수와 관련된 생리활성을 조사하기 위하여 뿌리조직으로부터 마이크로솜을 분리하였고, 마이크로솜 ATPase (이온점프) 활성을 분광학적 방법인 enzyme-coupled 분석방법에 따라 측정하였다. 마이크로솜 ATPase의 활성에 미치는 여러 가지 이온의 효과 또는 ATPase의 이온선택성을 조사하기 위하여 $10mM\;Na^+$과 $120mM\;K^+$을 포함하는 대조용액에서의 평균활성을 측정한 결과 190 nmol/min/mg protein으로 나타났다. 대조활성에 비하여 $Na^+$을 포함하지 않은 $130mM\;K^+$ 용액에서는 활성이 150%로 증가하였고, $K^+$을 포함하지 않은 $130mM\;Na^+$ 용액에서는 활성이 63%로 감소되었다. 반응용액의 $K^+$ 농도에 따른 활성변화를 측정한 결과, ATPase의 활성은 외부용액의 $K^+$ 농도 증가에 따라 활성이 증가됨을 알 수 있었다. 또한 마이크로솜 ATPase 활성은 반응용액의 pH 감소에 따라 증가되어 $pH\;6{\sim}7$에서는 비교적 높은 활성을 보였으나, pH 8 이상에서는 급격히 활성이 감소되었고, pH 9에서는 80%이상의 활성이 저해되었다. $Ca^{2+}$에 의한 이온펌프의 활성조절 여부를 평가하기 위해서 마이크로솜 내부 및 외부의 $Ca^{2+}$에 의한 ATPase 활성변화를 측정하였다. 마이크로솜 ATPase의 활성은 반응액의 $Ca^{2+}$ 농도가 낮아질수록 증가하여 $10^{-9}M$ 이하에서 최대활성이 관측되었고, $Ca^{2+}$ 농도가 증가할수록 활성은 감소하여 $500\;{\mu}M$ 전후에서 50%의 활성이 감소하였다. 또한 ATPase의 활성은 마이크로솜 내부의 $Ca^{2+}$ 농도증가에 의해서 저해되어, $Ca^{2+}\;ionophore\;A23187$처리에 의한 외부의 $Ca^{2+}$ 유입에 의해서 약30%의 활성감소를 보였으며, EGTA 처리에 의한 $Ca^{2+}\;chelation$에 의해서 마이크로솜 내부의 $Ca^{2+}$ 농도가 감소되었을 때, ATPase 활성은 증가하였다. 위의 조건에서 실제 마이크로솜 내부로의 $Ca^{2+}$ 유입 여부는 $‘Ca^{2+}’$를 이용하여 확인하였다. 이상의 결과는 마이크로솜 막에 위치한 ATPase의 내부 및 외부에 $Ca^{2+}$에 의한 효소활성 조절부위가 각각존재함을 시사한다. In order to investigate the mechanisms of epithelial ion transports, microsomes of soybean roots were prepared and the activity of microsomal ATPases was measured by an enzyme-coupled assay. The effects of various ions were evaluated on the total activity of microsomal ATPases and the average activity was 190 nmol/min/mg protein in the control solution containing $10\;mM\;Na^+\;and\;120\;mM\;K^+$. The activities were increased to 150% and decreased to 63% of the control activity in the solution containing $130\;mM\;K^+$ without $Na^+$ and in the solution containing $130\;mM{\;}Na^+$ without $K^+$, respectively. In general, the activity of microsomal ATPase was increased by$K^+$ in a concentration-dependent manner The activity was also increased at lower pH and relatively higher activities were observed in the pH range of $6{\sim}7$. However, the activity was decreased at weak alkaline $pH\;and{\sim}80%$ of the activity was inhibited at pH 9. Since intracellular $Ca^{2+}$ has been known to control the activity of various enzymes, we have investigated the effects of intra-and extrarnicrosomal $Ca^{2+}$ on the activity of microsomal ATPases. The maximal activity was obtained at the extrarnicrosomal $Ca^{2+}$ concentrations below 1 nM. The activity was gradually decreased by increasing $‘Ca^{2+}’$ concentration and 50% inhibition was observed at ${\sim}500{\;}{\mu}M{\;}Ca^{2+}$. The increase in luminal $Ca^{2+}$ concentration also inhibited the activity of microsomal ATPase. When the influx of external $Ca^{2+}$ was induced by $Ca^{2+}$ ionophore A23187 treatment, the activity was decreased by 30%; however, it was recovered by EGTA-induced chelation of $Ca^{2+}$. These results suggest that the presence of $Ca^{2+}$ regulation sites on both cytoplasmi and luminal sides of microsomal ATPases.

Hg2+ 에 의한 토마토 뿌리조직 마이크로솜 H+-ATPase 의 가역적 저해

김영기,조광현,신대섭 한국농화학회 1999 Applied Biological Chemistry (Appl Biol Chem) Vol.42 No.4

In order to characterize the effects of heavy metal ions on the microsomal ATPase activities, microsomes were prepared from the roots of tomato plant and the activity of microsomal ATPase was measured by an enzyme-coupled assay. Hg^(2+) inhibited the activity of microsomal ATPase in a dose-dependent manner, while Gd^(3+), Fe^(3+), La^(3+), Zn^(2+), and Pb^(2+) inhibited not only the ATPase activity but also the activities of enzymes used in the assay. However, Cs^+ and Ba^(2+) showed no significant effect. Hg^(2+) inhibited the activities of both plasma membrane and vacuolar membrane H^+-ATPases. In the dose-response to Hg^(2+), the activities of both microsomal H^+-ATPases were severely inhibited at the concentration of Hg^(2+) above 10 μM and were completely inhibited at 1 mM Hg^(2+). Apparent Ki values of Hg^(2+) on the inhibitions of plasma membrane and vacuolar membrane H^+-ATPases were 8 μM and 58 μM, respectively. The Hg^(2+)+-induced inhibitions were reversible since the addition of dithiothreitol completely reversed the inhibitory effects of Hg^(2+). These results suggest that the inhibitory effects of Hg^(2+) on both plasma, membrane and vacuolar membrane H^+-ATPases are nonselective and reversible.