• The Korean Journal of Pharmacology
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• v.12 no.1
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• pp.7-14
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• 1976

Aconiti tuber butanol fraction shows positive inotropic effect on the isolated atrium of rabbit heart. To investigate the mechanism, the effect on microsomal ATPase activity of rabbit heart is observed. The microsomal fraction which contains the $Na^+$- and $K^+$-activated ATPase in the presence of $Mg^{++}$ is isolated from the left ventricle of rabbit heart. The microsomal ATPase activity is maximally stimulated at $Na^+$ and $K^+$ concentration of 100 mM and 10 mM respectively. Microsomal $Na^+-K^+$-activated ATPase is inhibited by ouabain and Aconiti tuber butanol fraction. Ouabain and Aconiti tuber butanol fraction depress $Na^+$-stimulation on microsomal ATPase activity, and the inhibitory effects are not completely reversed at $Na^+$ concentration of 300 mM. Also, $K^+$-stimulation on microsomal ATPase activity is inhibited by ouabin and Aconiti tuber butanol fraction and the inhibitions are not compeletely reversed at $K^+$ concentration of 30 mM. It is, therefore, suggested that the inhibitory effect of Aconiti tuber butanol fraction on the microsomal ATPase activity may contribute to leading to the positive inotropic effect.

• The Korean Journal of Physiology
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• v.29 no.2
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• pp.269-277
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• 1995

Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was $329{\pm}10$ nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular $Ca^{2+}-ATPase$, inhibited about 50% of the activity, leaving $178{\pm}18\;nmol/min .mg$ protein (n=6), indicating that the $Ca^{2+}-ATPase$ is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in $^{45}Ca^{2+}$ uptake experiments. Inositol 1,4,5-trisphosphate $InsP_{3}, 4\;{\mu}M$, an agonist of $InsP_{3}$-sensitive $Ca^{2+}$ release channel ($InsP_{3}$, receptor), and Ca-ionophore A23187 $(10\;{\mu}M)$ induced $^{45}Ca^{2+}$ releases of 20% and 50% of stored $^{45}Ca^{2+}$, respectively. The addition of $(10\;{\mu}M\;InsP_{3}$ also increased the microsomal ATPase activity from $282{\pm}8$ nmol/min mg protein to $334{\pm}21$ nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ;$InsP_{3}-induced$ effects were blocked by either thapsigargin or heparin suggesting that: 1) the $InsP_{3}-induced$ increase in ATPase activity is mediated by microsomal $Ca^{2+}-ATPase$, and 2) dissipation of $Ca^{2+}$ gradient across the microsomal membrane is responsible for the $InsP_{3}-induced$ effect. In order to test the dependence of the $Ca^{2+}-ATPase$ activity on the activity of $InsP_{3}-induced$ the activity of ATPases was monitored in various concentrations of free $Ca^{2+}$ using $EGTA-Ca^{2+}$ buffers. The $Ca^{2+}$-dependent biphasic change is the well-known character of $InsP_{3} receptor but not of microsomal $Ca^{2+}-ATPase$ in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of $397{\pm}36nmol/min\;.mg$ protein was obtained in the solution containing 100 nM free $Ca^{2+}$. Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal $Ca^{2+}-ATPase$ to the $InsP_{3}$ receptors in epithelial microsomes.

• The Korean Journal of Pharmacology
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• v.2 no.2
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• pp.35-42
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• 1966

The author investigated the effect of $Mg^#$, $Ca^#$, $Na^+$, $K^+$ and creatine phosphate on the ATPase activity of microsomal fraction isolated from rabbit uterus and obtained the following results : 1) The uterine microsomal fraction contained the $Na^+-$ and $K^+-$ activated ATPase in the presence of $Mg^#$. The ATPase activity increased with protein content in the fraction. 2) The maximum ATPase activity was obtained at $Na^+$ and $K^+$ concentraction of 100 mM respectively. 3) In the absence of $Mg^#$, the ATPase was not activated by $Na^+$ and $K^+$, but inhibited. 4) Car stimulated the $Na^+-$ and $K^+-$ activated ATPase in the presence of $Mg^#$. However, in the absence of $Mg^#$, the ATPase was not activated by $Ca^#$. 5) The $K^+-$ activated ATPase activity was greater than the $Na^+-activated$ ATPase under all conditions. 6) The $Na^+-$ and $K^+$ activated ATPase activity was increased by addition of creatine phosphokinase and creatine phosphate to the reaction mixture.

• Korean Journal of Environmental Agriculture
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• v.20 no.1
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• pp.28-33
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• 2001

Lettuces were grown hydroponically in three different nutrient solutions, normal and 30 or 50 mM $KNO_3-added$ nutrient solutions, and the electrical conductivities of the nutrient solutions were 1.0, 4.5, and 6.5 dS/m, respectively. Lettuces grown in the $KNO_3-added$ nutrient solutions showed a decrease in the germination ratio and the lower indices of growth, such as plant height, stem diameter, leaf length, and leaf width. Microsomes were prepared from the roots of lettuce and characteristics of microsomal ATPases were investigated. The activities of microsomal ATPases grown in the 30 mM and 50 mM $KNO_3-added$ nutrient solutions were higher than that grown in the normal nutrient solution. The highest activities of microsomal ATPases were observed at pH 7.0 in all culture conditions. The activities of microsomal ATPases were increased in a reaction buffer solution containing high concentration of $K^+$, whereas they were decreased in a reaction buffer containing $Na^+$. The stimulating effect of $K^+$ in the reaction buffer was greater on the microsomal ATPases of lettuces grown in the $KNO_3-added$ nutrient solutions than that grown in the normal nutrient solution. These results imply that the activities of microsomal ATPases in the root tissue are increased as increasing the $KNO_3$ concentration in the hydroponical nutrient solution.

• Applied Biological Chemistry
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• v.41 no.2
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• pp.130-136
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• 1998

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 ${\sim}30%$ and ${\sim}38%$ of total microsomal ATPase activity by using their specific inhibitor, vanadate and nitrate $(NO^-_3)$, respectively. The inhibitory effects of vanadate and $NO^-_3$ were additive and the simultaneous additions of these two inhibitors decreased the total activity up to $50{\sim}70%$. 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 ${\sim}30%$ 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 $NO^-_3$ 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 $NO^-_3-sensitive$ 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, $NO^-_3$ 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 $Ca^{2+}$.

• The Korean Journal of Physiology
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• v.20 no.2
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• pp.257-270
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• 1986

It has been reported that the luteal function may be regulated by the intracellular calcium in luteal cells (Higuchi et al, 1976; Dorflinger et at, 1984; Gore and Behrman, 1984) which is adjusted partially by $Ca^{++}-ATPase$ activities in luteal cell membranes (Verma and Pennistion, 1981). However, the physicochemical and kinetic properties of $Ca^{++}-ATPase$ in luteal membranes were not fully characterized. This study was, therefore, undertaken to partially characterize the physicochemical and kinetic properties of $Ca^{++}-ATPase$ system in luteal membranes and microsomal fractions, known as an one of the major $Ca^{++}$ storge sites (Moore and Pastan, 1978), from the highly luteinized ovary Highly luteinized ovaries were obtained from PMSG-hCG injected immautre female rats. Light membrane and heavy membrane fractions and microsomal fractions were prepared by the differential and discontinuous sucrose density gradient centrifugation method desribed by Bramley and Ryan (1980). Light membrane and heavy membrane fractions and microsomal fractions from highly luteinized ovaries are composed of the two different kinds of $Ca^{++}-ATPase$ system. One is the high affinity $Ca^{++}-ATPase$ which is activated in low $Ca^{++}$ concentration (Km, 10-30 nM), the other is low affinity $Ca^{++}-ATPase$ activated in higher $Ca^{++}$ concentration $(K_{1/2},\;40\;{\mu}M)$. At certain $Ca^{++}$ concentrations, activities of high and low affinity $Ca^{++}-ATPase$ are the highest in light membrane fractions and are the lowest in microsomal fractions. It appeares that high affinity $Ca^{++}-ATPase$ system have 2 binding sites for ATP (Hill's coefficient; around 2 in all membrane fractions measured) and the positive cooperativity of ATP bindings obviously existed in each membrane fractions. The optimum pH for high affinity $Ca^{++}-ATPase$ activation is around S in all membrane fractions measured. The lipid phase transition temperature measured by Arrhenius plots of high affinity $Ca^{++}-ATPase$ activity is around $25^{\circ}C$. The activation energies of high affinity $Ca^{++}-ATPase$ below the transition temperature are similar in each membrane fractions, but at the above transition temperature, it is the hightest in heavy membrane fractions and the lowest in microsomal fractions. According to the above results, it is suggested that intracellular $Ca^{++}$ level, which may regulate the luteal function, may be adjusted primarily by the high affinity $Ca^{++}-ATPase$ system activated in intracellular $Ca^{++}$ concentration range $(below\;0.1\;{\mu}M)$.

• Korean Journal of Environmental Agriculture
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• v.21 no.2
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• pp.102-108
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• 2002

In order to investigate the mechanism of growth inhibition by salt stress, lettuces were grown hydroponically in three different nutrient solutions, normal and 30 mM or 50 mM $KNO_3$-added nutrient solutions, and the electrical conductivities of these solutions were 1.0, 4.5, and 6.5 dS/m, respectively. The activities of plasma and vacuolar $H^+$-ATPases in the root tissue of lettuce were measured by specific inhibitors, 100 ${\mu}M$ vanadate and 50 mM $NO_3^-$, respectively. Microsomal ATPase activity of lettuce grown in the normal nutrient solution was $356\pm1.5$ nmol/min/mg protein. When lettuces were grown in 30 mM and 50 mM $KNO_3$-added nutrient solutions, total activities of microsomal ATPases were increased by 1.6 and 1.9 times, respectively, and the increases were mainly mediated by vacuolar $H^+$-ATPase. These results show that lettuces adapt themselves to salt-stressed condition by increasing the activities of $H^+$-ATPases. Effects of various heavy metal ions were investigated on the microsomal ATPases and various metal ions at 100 $\mu M$ inhibited the activities by 10$\sim$25%. $Cu^{2+}$ showed the highest inhibitory effect on the vacuolar $H^+$-ATPase. These results suggest that lettuce increases the activities of root ATPases, specially that of vacuolar $H^+$-ATPase, in salt-stressed growth conditions and $Cu^{2+}$ could be a useful tool to control the activity of vacuolar $H^+$-ATPase.

• Applied Biological Chemistry
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• v.42 no.4
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• pp.298-303
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• 1999

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 $10\;{\mu}M$ 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 $80\;{\mu}M$ and $58\;{\mu}M$, 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.

• Applied Biological Chemistry
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• v.40 no.3
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• pp.202-208
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• 1997

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.

• The Korean Journal of Pharmacology
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• v.2 no.1 s.2
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• pp.31-40
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• 1966

The microsomal fraction is isolated from rabbit heart and skeletal muscle. The fraction is found to contain the $Na^+$-and $K^+$-activated ATPase. The maximal ATPase activity is obtained in $Na^+$ and $K^+$ concentration of 100 mM. Calcium itself stimulates the $Na^+$-and $K^+$-activated portion of ATPase in the presence of $Mg^{++}$. However, calcium does not stimulate ATPase in the absence of $Mg^{++}$.