• Applied Biological Chemistry
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• v.48 no.3
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• pp.212-216
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• 2005

Thapsigargin is a specific antagonist of SR/ER-type $Ca^{2+}-ATPase$ in animal tissue, and it was used to characterize the microsomal ATPases prepared from the roots of tomato. When $10\;{\mu}M$ thapsigargin was added, it inhibited the microsomal ATPase activity by 30%. The thapsigargin-induced inhibition was dose-dependent. Since the activity of $Ca^{2+}-ATPase$ is very low in the roots of tomato tissue, it is possible that thapsigargin inhibits the activities of major $H^+-ATPases$ located in plasma and vacuolar membranes. The inhibitory effect of thapsigargin was reduced when the vacuolar $H^+-ATPase$ activity was inhibited by ${NO_3}^-$. However, the effect of thapsigargin was not observed on the $H^+-ATPase$ activity located in the plasma membrane. These results suggest that thapsigargin inhibits the vacuolar $H^+-ATPase$ activity in the roots of tomato.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.441-448
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• 2000

The production and cation flux-mediated activation of the P-type ATPase in Helicobacter pylori was investigated. Using the polymerase chain reaction (PCR), the proton pump genotype of H. pylori was found to be positive for both F-type and P-type ATPases. Yet, their production in terms of enzyme specific activity varied substantially depending on H. pylori strains, ranging over 3-fold. Its main constituent appeared to be the P-type ATPase pool, in contrast to other common bacterial compositions. Interestingly, the F-type ATPase was observed only when intact H. pyloricells were exposed to pH 4.5 or above (37$^{\circ}C$ for 1 h). In contrast, significant amounts of the P-type ATPase still remained after 1 h of cell treatment even at pH below 4.5. By enriching the acidic medium with RPMI(pH 3.0), the P-type ATPase was stabilized, accompained by inactivation of the F-type ATPase. Using H. pylori membrane vesicles, it was found that ammionia-mediated cation flux increased the rate of ATP hydrolysis by the P-type ATPase. Accordingly, these data strongly suggest that the P-type ATPase is involved or functions as an effective regulator for the cation flux across the H. pylori membrane, thereby reducing the risk of excess proton influx.

• Applied Biological Chemistry
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• v.44 no.2
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• pp.67-72
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• 2001

$H^+-ATPase$ located on plasma and vacuolar membranes play major roles in various cellular physiological processes. In order to investigate the physiological roles of $H^+-ATPase$, microsomes were prepared from tomato roots and the effects of various anions were measured on the activities of $H^+-ATPase$. $H^+-ATPase$ was inhibited by various anions. Citrate and phosphate were chosen to investigate detailed inhibitory mechanisms on $H^+-ATPase$ since they showed different levels of inhibition. Inhibitory effect of citrate was observed at the concentrations above 3 mM. When 20 mM citrate was added, the ATPase activity was decreased by 50-60%. However, the inhibitory effect of citrate was decreased by increasing the concentration of$Mg^{2+}$ The citrate-induced inhibited activity was recovered by the addition of $Mg^{2+}$ Addition of 7 mM $Mg^{2+}$ completely removed the inhibitory effect of citrate and the activity recovered to the level of the control experiment. These results imply that citrate chelates $Mg^{2+}$ and thus inhibits $H^+-ATPase$. Meanwhile, the inhibitory effect of phosphate was observed at the concentration above 3 mM and the activity was decreased by 50% in the presence of 30 mM phosphate. Further addition of $Mg^{2+}$ showed no recovery on the activity. These results imply that the inhibitory effect of phosphate is not dependent upon the concentration of $Mg^{2+}$.

• Applied Biological Chemistry
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• v.46 no.2
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• pp.84-89
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• 2003

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 $H^+-ATPases$.

• The Korean Journal of Zoology
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• v.17 no.2
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• pp.93-102
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• 1974

Fragmente dsarcoplasmic reticulum of rabbit skeletal muscle was prepared and biochemical properties of its ATPase activity were studied. The ATPase of the fragments could be distinguished as $Mg^++ - ATPase and (Mg^++ - Ca^++)$-ATPase. The activity of $(Mg^++ - Ca^++)$-ATPase was predominant over that of $Mg^++$-ATPase in the temperature range of $0 \\sim 40^\\circ C$ and in the pH 6.4$\\sim$7.6. At higher temperatures the predominance of $(Mg^++ - Ca^++)$-ATpase was far greater. The apparent energies of activation were 14 kcal/mole for $Mg^++$-ATPase, 21kcal/mole for $(Mg^++ - Ca^++)$-ATPase, and 18kcal/mole for total ATPase. Changes in pH and Mg concentration did not alter the energies of activation of these ATPases. The Km values of these ATPases were found to be 0.36 mM for $Mg^++$-ATPase, 2.20 mM for $(Mg^++ - Ca^++)$-ATpase, and 0.86 mM for total ATPase.

• 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.

• KSBB Journal
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• v.17 no.1
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• pp.59-62
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• 2002

The enzyme sensor for ATPase activity consisted of an immobilized membrane of two enzymes, purine nucloside phosphorylase (NP) and xanthine oxidase (XOD), and oxygen electrode. The $H^ +-ATPase $ rate of the plasma membranes increased by low root temperature. A cucumber and a pumpkin plasma membrane $H^ +-ATPase $$ activities measured by the proposed sensor system were in good agreement with the results obtained by a conventional UV spetrometer assay. One cycle of assay could be completed within 3 minutes.

• Biomolecules & Therapeutics
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• v.5 no.3
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• pp.228-232
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• 1997

AU-164 was synthesized as a reversible gastric $H^+/K^+$ ATPase inhibitor, and its effects were tested in various systems. AU-164 inhibited rabbit gastric $H^+/K^+$ ATPase with an $IC_{50}$/ of 9 $\mu$M. On the other hand, AU-164 was a weak inhibitor for dog kidney $Na^+/K^+$ ATPasc, indicating the selectivity for gastric $H^+/K^+$ ATPase. The reversible property of the AU-164-induced inhibition of $H^+/K^+$ ATPase was confirmed by filtering the inhibition mixture through Sephadex G-25M column. In vivo basal acid secretion was also inhibited by AU-164 under the pylorus ligation of Sprague-Dawley rats. In addition, AU-164 protected dose dependently gastric lesion induced by ethanol in rats. The $ED_{50}$ value of 62 mg/kg p.o was estimated. These results suggest that AU-164 is a potent, selective and reversible gastric $H^+/K^+$ ATPase inhibitor, and that AU-164 has a potential use for the clinical therapeutics of peptic ulcer disease.

• 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 $Ca^{2+}$.

• Applied Biological Chemistry
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• v.45 no.2
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• pp.53-58
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• 2002

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.