• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.3
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• pp.192-198
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• 2003

BiP, immunoglobulin binding protein, is an ER homologue of Hsp70. However, unlit other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demo strafed the presence of potential regulatory proteins for BiP using a pull -down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull -down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein, light proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp170 and BiP where identified. while the other were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for BiP was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.

• The Korean Journal of Nuclear Medicine
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• v.32 no.2
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• pp.121-128
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• 1998

Purpose: $Na^+-K^+$ ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristisc of T1-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive $Na^+-K^+$ ATPase activity using T1-201 and compare with that using Rb-86. Materials and Methods: Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular $Na^+-K^+$ ATPase activity. $Na^+-K^+$ ATPase activity was measured as a percentage decrease in cellular uptake of T1-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Results: $Na^+-K^+$ ATPase activity measured with T1-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 10-70% or 28% increases from baseline activity, respectively. Conclusion: These results suggests that 71-201 could replace Rb-86 in measurement of ouabain sensitive $Na^+-K^+$ ATPase activity in vitro. High level of glucose concentration decreased cellular $Na^+-K^+$ ATPase activity, but insulin or PMA increased it.

• YAKHAK HOEJI
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• v.29 no.2
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• pp.76-89
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• 1985

The effects of ginseng saponin on the activity, phosphorylation, [$^{3}$H] ouabain binding and light scattering (disruption) of purified $Na^{+}$ ,$K^{+}$ -ATPase isolated from the outer medulla of sheep kidney were compared to those of gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 on the same parameters. $Na^{+}$ , $K^{+}$ -ATPase activity, phosphorylation, and [$^{3}H$] ouabain binding were inhibited by ginseng saponin (triol>total>diol), SDS, or Triton X-100, but increased by gypsophila saponin. Low doses of ginseng saponin (3.mu.g saponin/.mu.g protein) decreased phosphorylation sites and ouabain binding site concentration (Bmax) without any change of turnover number and affinity for ouabain binding which were decreased by high dose of ginseng saponin (over 10.mu.g saponin/.mu.g protein), SDS or Triton X-100. On the other hand, gypsophila saponin increased the affinity without any change of Bmax for ouabain binding. Inhibition of $Na^{+}$ ,$K^{+}$ -ATPase activity by ginseng saponin and SDS or Triton X-100 appeared before and after decrease in light scattering, respectively. These data suggest that ginseng saponins (total, diol, triol saponin) inhibit $Na^{+}$ , $K^{+}$ -ATPase activity by specific direct and general detergent action at low and high concentrations, respectively, and this inhibitory action of ginseng sapornin to $Na^{+}$ , $K^{+}$ -ATPase is not general action of all saponins.

• The Korean Journal of Physiology
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• v.8 no.1
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• pp.55-65
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• 1974

The effect of ginseng on the ATPase activity of rabbit ref cell membrane has been investigated. The experiments were also designed to determine whether the components of ginseng could be attributed to the effect on ATPase activity which dependent upon sodium plus potassium and is sensitive to ouabain. The following results were observed. 1. The activity of the $Na^{+}-K^{+}-ATPase$ from red cell membrane is stimulated by ginseng, and the concentration of ginseng for half-maximal activity is about 15 mg%. The pH optimum for the ginseng sensitive component is 7.6. 2. The portion of the enzyme activity stimulated by ginseng is completely abolished by ouabain. 3. The activating effect of ginseng on the ATPase, with a given concentration of sodium in the medium, is increased by raising the potassium concentration but activity ratio is decreased. 4. The activating effect of ginseng on the ATPase, with a given concentration of potassium in the medium, is increased by raising the sodium concentration but the activity ratio is decreased. 5. The ATPase activity is increased by small amounts of calcium but inhibited by larger amounts and the rate of activity by ginseng is constant. 6. The action of ginseng on the ATPase activity was not related to the sulfhydryl group of cysteine, the amino group of lysine, the imidazole group of histidine, the quanidinium group of arginine, the carboxyl group of aspartic acid, or the hydroxyl group of threonine. 7. The activating effect of ginseng on the ATPase activity may be not due to a saponin which is contained in ginseng.

• 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 $(below\;0.1\;{\mu}M)$.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.9-11
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• 2002

Plasma membrane Na$^{＋}$-K$^{＋}$ ATPase (pump) is an essential component to maintain asymmetrical ion distribution across cell membrane. The Na$^{＋}$-K$^{＋}$ ATPase was discovered by Jens C. Skou in 1957 and since then physiological and biochemical properties of the enzyme have been extensively studied. Jens C. Skou was awarded the 1997 Nobel Prize in chemistry for his discovery of the Na $^{＋}$ - $K^{＋}$ ATPase.(omitted)

• Bulletin of the Korean Chemical Society
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• v.29 no.9
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• pp.1717-1722
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• 2008

The 70 kDa heat-shock protein (Hsp70) involved in various cellular functions, such as protein folding, translocation and degradation, regulates apoptosis in cancer cells. Recently, it has been reported that the green tea flavonoid (−)-epigallocatechin 3-gallate (EGCG) induces apoptosis in numerous cancer cell lines and could inhibit the anti-apoptotic effect of human Hsp70 ATPase domain (hATPase). In the present study, docking model between EGCG and hATPase was determined using automated docking study. Epi-gallo moiety in EGCG participated in hydrogen bonds with side chain of K71 and T204, and has metal chelating interaction with hATPase. Hydroxyl group of catechin moiety also participated in metal chelating hydrogen bond. Gallate moiety had two hydrogen bondings with side chains of E268 and K271, and hydrophobic interaction with Y15. Based on this docking model, we determined two pharmacophore maps consisted of six or seven features, including three or four hydrogen bonding acceptors, two hydrogen bonding donors, and one lipophilic. We searched a flavonoid database including 23 naturally occurring flavonoids and 10 polyphenolic flavonoids with two maps, and myricetin and GC were hit by map I. Three hydroxyl groups of B-ring in myricetin and gallo moiety of GC formed important hydrogen bonds with hATPase. 7-OH of A-ring in myricetin and OH group of catechin moiety in GC are hydrogen bond donors similar to gallate moiety in EGCG. From these results, it can be proposed that myricetin and GC can be potent inhibitors of hATPase. This study will be helpful to understand the mechanism of inhibition of hATPase by EGCG and give insights to develop potent inhibitors of hATPase.

• 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 Journal of Internal Korean Medicine
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• v.19 no.1
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• pp.431-441
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• 1998

This study was undertaken to determine whether Buthus exract(BTE) affects Na^+-K^+-ATPase$ activity of nervous tissues. The enzym activity was measured in synaptosomal fraction prepared from rabbit brain cortex. Na^+-K^+-ATPase$ activity was inhibited by BTE over concentration range of 0.05-0.5% in a dose-dependent manner. The enzyme activity was increased by an increase in $Na^+$ concentration from 5 to 100mM, $K^+$ concentration from 0.5 to 10mM, and $Mg^{2+}$ concentration from 0.2 to 5mM. These changes in ion concentrations did not produce any effect on the inhibitory effect of BTE on $Na^+-K^+-ATPase$ activity. An increase in ATP concentration from 0.1 to 3mM caused an increase in the enzyme activity. The inhibition of the enzyme activity by BTE were not different between two ATP concentrations. A sulfhydryl group protector DTT prevented PCMB-induced inhibition of $Na^+-K^+-ATPase$ activity, but the BTE-induced inhibition was not altered by DTT. The inhibition of enzyme activity by combination of ouabain and BTE was not different from that by Buthus alone. These results suggest that Buthus exerts inhibitory effect on $Na^+-K^+-ATPase$ activity in cerebral synaptosomes, and the action mechansim is similar to that of ouabain.

• Applied Microscopy
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• v.40 no.4
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• pp.261-266
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• 2010

ATP is the energy source synthesized at the electron transferase that consist of complex I, II, III, IV and V in mitochondrial cristae. The complex V functions as ATPase which composed of sub-complex $F_0$ and $F_1$. Porin or VDAC (voltagedependent anion-selective channel), is a family of small pore-forming proteins of the mitochondrial outer membrane, and play important roles in the regulated flux of anion, proton and metabolites between the cytosolic and mitochondrial compartments. The channel allows the diffusion of negatively charged solutes such as succinate, malate, and ATP in the fully open state, but of positively charged ions in subconducting state. In this study, in order to investigate the relationship of the function and localization between porin and ATPase we observed the distribution of porin and ATPase in the mitochondria of the bovine heart. Monoclonal antibodies against porin and ATPase ${\beta}$-subunit were used to detect porin and ATPase using light microscope with immunohistochemistry and immunofluorescence, and using electron microscope with immunogold-labeling. ATPase were stained in longitudinal section region in cardiac muscle, porin were stained in longitudinal section region in cardiac muscle. We viewed more specific pattern of localization and distribution of these proteins using immunofluorescence method. There were some region which were labeled with porin or ATPase respectively, and others which were labeled both proteins in cardiac muscle. The electron microscope results showed that immunogold labeled porin were labeled locally at mitochondrial outer membrane and ATPase were labeled evenly at mitochondrial cristae. But ATPase was not labeled at mitochondria cristae. These results confirmed the subcellular localizations of porin and ATPase in mitochondrial outer membrane and cristae. Also, we assumed that ATP synthesis always does not activation in all mitochondria exist in the bovine cardiac muscle.