• Bulletin of the Korean Chemical Society
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• v.30 no.8
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• pp.1724-1728
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• 2009

Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that $Mg^{2+}$ stimulated the ATPase activity with the highest rate and $Mn^{2+}$ with about 40% rate as compared to the $Mg^{2+}$.

• Applied Microscopy
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• v.18 no.2
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• pp.187-204
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• 1988

The present experiment was performed to investigate the acute and subacute effect of lead acetate on ultrastructural and biochemical changes in mouse diencephalon. In acute case, mouse were peritoneally injected with lead acetate at a dose of 0.26 mmole/kg body weight, and after treatment, mouse were sacrificed at time intervals of 12, 24, 48, and 96 hours. In subacute case, mouse were injected at doses of 0.07 mmoie/kg B. W. and 0.13 mmole/kg B.W. once at two days, and after treatment, mouse wee sacrificed at 1 week, 2 weeks, and 3 weeks. It was observed that after acute treatment, changes composed of increased monoamine oxidase activity, $Na^{+}-K^{+}$ ATPase activity, decreased $Mg^{2+}$-APTase activity, wrinkled myelin, swollen Golgi apparatus and more dense synaptic vesicle in nerve terminal. After subacute treatment, decreased monoamine oxidase activity, increased $Mg^{2+}$-ATPase, $Na^{+}-K^{+}$ ATPase, lose of myelin, uneven mitochondrial distribution, synaptic vesicular density and edema, but at a higher dose the effect was more severe. Therefore, lead acetate caused abnormal change of diencephalon, and at a subacute, it appears metal accumulative toxicity.

• The Korean Journal of Physiology
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• v.29 no.1
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• pp.69-80
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• 1995

Renal proximal tubular hypertrophy and hyperfunction are known to be early manifestations of experimental and human diabetes. As the hypertrophy and hyperfunction have been suggested to be central components in the progression to renal failure, an understanding of their underlying causes is potentially important for the development of therapy. A primary rabbit kidney proximal tubule cell culture system was utilized to evaluate the possibility that the renal proximal tubular hypertrophy and hyperfunction observed in vivo in diabetes mellitus, can be attributed to effects of elevated glucose levels on membrane transport systems. Primary cultures of rabbit proximal tubules, which achieved confluence at 10 days, exhibited brush-border characteristics typical of proximal tubular cells. Northern analysis indicated $2.2{\sim}2.3$ and 2.0 kb Na/glucose cotransporter RNA species appeared in fresh and cultured proximal tubule cells after confluence, repectively. The cultured cells showed reduced Na/glucose cotransporter activity compared to fresh proximal tubules. Primary cultured proximal tubule cells incubated in medium containing 20 mM glucose have reduced ${\alpha}-MG$ transport compared to cells grown in 5 mM glucose. In the proximal tubule cultures incubated in medium containing 5 mM or 20 mM glucose, phlorizin at 0.5 mM inhibited 0.5 mM ${\alpha}-MG$ uptake by 84.35% or 91.85%, respectively. The uptake of 0.5 mM ${\alpha}-MG$ was similarly inhibited by 0.1 mM ouabain (41.97% or 48.03% inhibition was observed, respectively). In addition, ${\alpha}-MG$ uptake was inhibited to a greater extent when $Na^{+}$ was omitted from the uptake buffer (81.86% or 86.73% inhibition was observed, respectively). In cell homogenates derived from the primary cells grown in 5 mM glucose medium, the specific activity of the Na/K-ATPase $(6.17{\pm}1.27\;{\mu}mole\;Pi/mg\;protein/hr)$ was 1.56 fold lower than the values in cell homogenates treated with 360 mg/dl D-glucose, 20 mM $(9.67{\pm}1.22\;{\mu}mole\;Pi/mg\;protein/hr)$. Total $Rb^{+}$ uptake occurred at a significantly higher rate (1.60 fold increase) in primary cultured rabbit kidney proximal tubule cell monolayers incubated in 20 mM glucose medium $(10.48{\pm}2.45\;nM/mg\;protein/min)$ as compared with parallel cultures in 5 mM glucose medium. $Rb^{+}$ uptake rate in 5 mM glucose medium was reduced by 28% when the cultures were incubated with 1 mM ouabain. The increase of the $Rb^{+}$ uptake by rabbit kidney proximal tubule cells in 20 mM glucose could be attributed primarily to an increase in the rate of ouabain-sensitive $Rb^{+}$ uptake $(5\;mM\;to\;20\;mM;\;4.68{\pm}0.85\;to\;8.38{\pm}1.37\;nM/mg\;protein/min)$. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Nafglucose cotransport system is inhibited.

• Development and Reproduction
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• v.24 no.3
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• pp.215-224
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• 2020

Seawater adaptability of steelhead trout increases along with the increase in the size of the fish, independent of parr-smolt transformation. Three 96 h seawater challenge tests were conducted to determine the size at which seawater adaptability of steelhead trout develops. Plasma Na⁺ and Cl^- levels, moisture content, gill Na⁺/K⁺ ATPase activity, and mortality during the 96 h after direct transfer to seawater (32 ppt) were determined. Plasma Na⁺ and Cl^- levels in 50 g fish continuously increased during the 96 h after the transfer to seawater (p<0.05), but the levels in 100 and 150 g fish leveled off after 24 h (p<0.05). Both 100 and 150 g size steelhead trout maintained muscle moisture content (%) better than 50 g size fish (p<0.05). Gill Na⁺/K⁺ ATPase activity in the 100 g size group increased in a time-dependent manner after transfer to seawater (p<0.05), whereas activity in the 50 and 150 g sizes did not increase (p>0.05), for which a possible explanation was discussed. A mere 2.6% mortality in both the 50 and 150 g size groups was observed. In conclusion, the current results indicate that 50 g size steelhead trout did not show development of a high level of hypoosmoregulatory capacity, whereas fish in the 100 and 150 g size groups showed a high level in our experimental conditions. Therefore, the steelhead trout larger than a 100 g size is recommended for transfer to seawater culture.

• YAKHAK HOEJI
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• v.30 no.1
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• pp.31-41
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• 1986

Sarcolemmal membrane fraction from canine ventricle was isolated from the discarded pellet after the first homogenization in the isolation procedure of sarcoplasmic reticulum (Method 1) and the protein yield, purity, and sidedness of this preparation were compared to those of sarcolemmal fraction prepared by method of Lee et al. (Method 2) and a slight modification of original protocol of Jones et al. (Method 3). Method 1 differed from Method 2 essentially only in that vigorous homogenization was carried out by omnimixer and homogenization medium containing 30mM Tris-maleate was used in the first step. The sarcolemmal fraction was enriched from 45 to 50 and 29-fold in [$^3H$] ouabain, [$^3H$] DHA, [$^3H$] QNB binding and $Na^+$, $K^+$-ATPase activity, respectively, compared to homogenate. Total $Na^+$, $K^+$-ATPase activity of highly sarcolemma enriched fraction was 144.6$\pm$16.4$\mu\textrm{mol}$ Pi/mg protein/hr, which was about 85%, of total ATPase activity, and the yield of the preparation was 15.7 mg protein per 100g of starting ventricular tissue. The sarcolemmal preparation supported $^{45}Ca^{2+}$-uptake in the presence of ATP but this uptake was not dependent on oxalate. Sarcolemmal $Na^+$, $K^+$-ATPase activity and detectable [$^3H$] ouabain binding were increased about 32% and 35%, respectively, by pretreatment of sarcolemmal fraction with optimal concentration of sodium dodecylsulfate (0.3-0.4mg/mg protein), suggesting that this preparation contained about 24% of sealed rightside-out vesicles, 26% of sealed inside-out vesicles, and 5001o of freely permeable (leaky) form. This procedure showed the highest protein yield and leaky population, compared to Method 2 and 3. On the other hand, sarcolemmal fraction prepared by Method 2 and 3 showed low value in protein yield but comtained high population of inside-out (46%) and rightside-out (49%) vesicles, respectively, compared to present procedure (Method 1). The results indicate that vigorous homogenization decreases the population of sealed sarcolemmal vesicles but increases the sarcolemmal protein yield per gram tissue and that this procedure is available for further purification of sarcolemmal fraction and for the receptor binding study of sarcolemma.

• The Korean Journal of Physiology
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• v.10 no.1
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• pp.35-40
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• 1976

In an attempt to examine the effect of ginseng on sodium transport across the biological membrane, we have studied effects of ginseng alcohol extract on the short-circuit current(SCC) and the $Na^+-K^+$-activated ATPase activity in isolated frog skin preparations. 1. Ginseng alcohol extract applied to the mucosal surface of the frog skin significantly increased SCC at low concentration($1{\sim}10mg%$) but decreased SCC at higher concentration($50{\sim}250mg%$). 2. Similarly, when the drug was added to the serosal bathing medium, the SCC was stimulated at low doses($5{\sim}25mg%$) and inhibibited at high doses($50{\sim}250mg%$). 3. $Na^+-K^+$-activated ATPase activity of the frog skin epidermal homogenate was significantly inhibited by ginseng alcohol extract, the effect being proportional to the concentration of the drug in the incubation mixture. These results may suggest that a low dose of ginseng alcohol extrat enhances the transepithelial sodium transport probably by increasing the permeability of outer membrane of the transporting cell to sodium ion, whereas a high dose of drug reduces the sodium transport primarly by inhibiting $Na^+-K^+$ ATPase mediated active transport step.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.4
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• pp.271-279
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• 1984

Effects of temperature and additives on the stability of actomyosin extracted from skeletal muscle of Israeli carp, Cyprinus carpio nudus, were studied by analyzing free SH-group, ATP-sensitivity and Ca-ATPase activity. The used additives were sucrose, sorbitol, Na-glutamate and L-cysteine. Furthermore, the denaturation constant($K_D$), protective effect(${\Delta}E/M$) and the other thermo-dynamic parameters on protein denaturation are systematically discussed. The actomyosin showed $4.12{\sim}4.68 mg/ml$ in protein concentration, $2.63{\sim}2.93\%$ in ribonucleic acid to the protein, $1:2.20{\sim}2.63$ in the binding ratio of myosin and actin, $4.33{\sim}5.26\%$ in fat content, 109.78 in ATP-sonsitivity, $0.159{\sim}0.201\;{\mu}M-Pi/min/mg-protein$ in Ca-ATPase activity and $3.3{\sim}3.4M/10^5$g-protein in free SH-group content. The first-order rate plots were obtained on the decrease of Ca-ATPase activity and ATP-sensitivity with an increase in temperature, while the free SH-group was increased to $60^{\circ}C$ and decreased rapidly above the temperature. The half-life of Ca-ATPase activity on the actomyosin Ca-ATPase was 280 min at $12^{\circ}C$, 125 min at $20^{\circ}C$, 55 min at $30^{\circ}C$ and 13 min at $40^{\circ}C$, and activation energy, activation enthalpy, activation entropy and free energy of the proteins at $20^{\circ}C$ wene 5,395 cal/mole, 4,814 cal/mole, -40.42 e.u. and 17,626 cal/mole, respectively. The protective effect of the additives on the actomyosin Ca-ATPase showed that the most effective material is $3\%$ sorbitol and followed in the order of $8\%$ Na-glutamate, $1\%$ sucrose and $1\%$ L-cysteine. The actomyosin was more stable at $-30^{\circ}C$ than at $0^{\circ}C$ and $-20^{\circ}C$. and when the additives were used in the low temperature storage, $8\%$ Na-glutamate was the most effective. $3\%$ sorbitol, $1\%$ sucrose and $1\%$ L-cysteine was to become lower in the order.

• The Korea Journal of Herbology
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• v.30 no.5
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• pp.15-21
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• 2015

Objectives : Methicillin-resistantStaphylococcus aureus(MRSA) is a human pathogen. New antibacterial agents are needed to treat MRSA-related infections. This study investigated the antibacterial activity of EtOH 70% extracts ofTonghyeonipal-dan(THD) which prescription is composed of oriental medicine against MRSA.Methods : The antibacterial activity of THD was evaluated against MRSA strains by using the Disc diffusion method, broth microdilution method, Checkerboard dilution test, and Time-kill test; its mechanism of action was investigated by bacteriolysis, detergent or ATPase inhibitors were used.Results : The minimum inhibitory concentration (MIC) of THD is 1,000~2,000 μg/mL against MRSA. In the checkerboard dilution test, fractional inhibitory concentration index (FICI) of THD in combination with antibiotics indicated synergy or partial synergism againstS. aureus. Furthermore, a time-kill assay showed that the growth of the tasted bacteria was considerably inhibited after 24 h of treatment with the combination of THD with selected antibiotics. For measurement of cell membrane permeability, THD 500 μg/mL along with concentration of Triton X-100 (TX) and Tris-(hydroxymethyl) aminomethane (TRIS) were used. In the other hand, N,N-dicyclohexylcarbodimide (DCCD) and Sodium azide (NaN3) were used as an inhibitor of ATPase. TX, TRIS, DCCD and NaN3 cooperation againstS. aureusshowed synergistic action.Conclusions : Accordingly, antimicrobial activity of THD was affected by cell membrane and inhibitor of ATPase were assessed. These results suggest that THD has antibacterial activity, and that THD extract offers great potential as a natural antibiotic against MRSA.

• Journal of Preventive Medicine and Public Health
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• v.23 no.4 s.32
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• pp.371-390
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• 1990

Production of free radicals of superoxide anion in tissues by cadmium, activities of superoxide dismutase and catalase to protect tissue damages caused by the free radicals and ATPase that plays an important role in energy metabolism at cellular level were investigated. Experiments in vivo were conducted with liver, kidney and testicle tissue homogenates of rats adding $0.05{\sim}0.50mM$ cadmium chloride, and in vivo experiments administering single dose of 5 mg of cadmium/kg of body weight in 0.1% cadmium chloride solution intraperitoneally 48 hours prior to evisceration. Production of superoxide radicals in liver and testicle increased with addition of cadmium in vitro, but not in kidney. In vivo experiments, however, superoxide radicals slightly increased in liver and kidney but not in testicle. Superoxide dismutase (Cu, Zn-SOD and Mn-SOD), catalase and ATPase (total, $Mg^{++}-\;&\;Na^+,\;K^+-$) activity decreased in the presence of cadimium in dose dependent manner. Reduction of these enzyme activities varied not only with dosage of cadmium but also with type of tissue and between in vitro and in vivo experiment.

• The Korean Journal of Physiology
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• v.17 no.2
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.