• 한국동물학회지
• /
• 제17권2호
• /
• pp.93-102
• /
• 1974

토끼의 골격근 小胞體 切片을 遠心分離하여 그 ATPase 活性의 生化學的性質을 $Mg^++ - ATPase 와 (Mg^++ - Ca^++)$-ATPase로 구분하여 조사하였다. $(Mg^++ - Ca^++)$-ATPase의 活性은 $0^\\circ - 40^\\circ C$의 범위, 그리고 pH 6.4-7.6의 범위에서는 $Mg^++$-ATPase보다 훨씬 높다. 이 현상은 온도가 높을수록 더욱 현저하다. 活性化에너지는 온도 $0^\\circ -40^\\circ C$의 범위에서는 $Mg^++$-ATPase가 14kcal/mole, $(Mg^++ - Ca^++)$-ATpase 가 21kcal/mole, 그리고 total ATPase 가 18kcal/mole 이다. 이 活性化에너지의 값은 pH와 Mg 濃度에 무관하다. 이들 효소의 Km의 값은 $Mg^++$-ATPase 가 0.36mM , $(Mg^++ - Ca^++)$-ATpase가 2.20mM, 그리고 total ATPaserk 0.86 mM이다.

• 약학회지
• /
• 제22권3호
• /
• pp.120-127
• /
• 1978

We have studied the effect of ouabain, tool ginseng saponin, panax saponin C (protopanaxatriol derivative) and ginsenoside $Rb_{1}$ (protopanaxadiol derivative) on $Na^+,K^+$-ATPase and $Mg^{++}$-ATPase activities were determined by the method of Robinson and ATPase activities were determined by the method of King. The $Na^+,K^+$-ATPase activities were inhibitied by 90.1% and 51.1% respectively at the concentration of $10^{-3}M$ and $10^{-4}M$ ouabain. The results are consistent with those of Robinson. The $Na^+,K^+$-ATPase activities were increased by 14.3% and 10.0% respectively at the concentration of $10^{-4}$g/ml and $10^{-5}$g/ml total ginseng saponin. Panax saponin C obtained by the method of Han and ginsenoside $Rb_{1}$ obtained by the method of Shibata were used. The $Na^+,K^+$-ATPase activities were increased in the presence of panax saponin C and the increased activity with panax saponin C was greater than that with total ginseng saponin. On the other hand ginsenoside $Rb_{1}$ showed an inhibitory effect on $Na^+,K^+$-ATPase. Total ginseng saponin, panax saponin C and ginsenoside $Rb_{1}$ had no effect on $Mg^{++}$-ATPase. Therefore, it may be concluded that total ginseng saponin has dual effects on microsomal $Na^+,K^+$-ATPase, that is, panax saponin C exhibits stimulatory action, whereas ginsenoside $Rb_{1}$ shows inhibitory action.

• Environmental Analysis Health and Toxicology
• /
• 제8권1_2호
• /
• pp.37-58
• /
• 1993

In order to investigate the toxicities of S-bioallethrin (5-biol) and its combination treatment with N-octyl bicycloheptene dicarboximide (MGK-264), the acute and subacute toxicity, and enzyme activity test were performed. $LD_{50}$ levels of S-biol and MGK-264 in rats are 640 mg/kg and 3, 280 mg/kg respectively. However, when rats were treated with the mixture of S-biol and MGK-264 (1 : 5 ratio), the $LD_{50}$ was decreased to 545 mg/kg. In serological analysis, ALT and LDH were increased in animals treated with the mixture. Also glucose level was significantly increased after 5 weeks in animals treated with both S-biol and the mixture. Other biochemical parameters such as cytochrome P-450 and NADPH-cytochrome c reductase in the liver and kidney were shown to be not significantly changed. Levels of total ATPase and $mg^{2+}$ ATPase were significantly decreased in the liver of animals treated with the mixture after 4-5 weeks. In addition, S-biol can alone decrease total ATPase activity. Total ATPase activity was also significantly decreased in the kidney after 5 week treatment with the mixture. Similarily, glucose-6-phosphatase activity was significantly decreased in animals treated with the mixture. When either S-biol or MGK-264 was administered, cholinesterase and carboxyesterase activities were slightly decreased but they were significantly decreased when the mixture was administered.

• Journal of Preventive Medicine and Public Health
• /
• 제23권4호
• /
• pp.371-390
• /
• 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.

• 대한약리학회지
• /
• 제18권2호
• /
• pp.69-77
• /
• 1982

$Na^+,\;K^+-ATPase$ is a component of plasma membrane in almost all animal cell, and maintains ionic distribution and membrane potential of normal cell. In the mechanism of adrenergic transmission, it is relatively well known that drug-receptor combination leads to stimulate adenylate cyclase and so on. In the cholinergic transmisison, the mechanism is not well known but is simply interpreted as the change of membrane permeability results from acetylcholine receptor interaction. To study the relationship between cholinergic transmission and membrane $Na^+,\;K^+-ATPase$, the effect of carbachol on $Na^+,\;K^+-ATPase$ activity in rabbit erythrocyte membrane is studied. The results are summarized as follows. 1) Total ATPase, $Mg^{+2}-ATPase$ and $Na^+,\;K^+-ATPase$ of rabbit erythrocyte membrane show maximum activities at 1mM of tris-ATP. 2) Total ATPase activity tends to increase when treated with carbachol $(10-^{-9}M-10^{-3}M)$. 3) The $Mg^{+2}-ATPase$ activity also tends to increase when treated with carbachol $(10-^{-9}M-10^{-3}M)$. 4) The $Na^+,\;K^+-ATPase$ activity is inhibited when treated with carbachol $(10-^{-9}M-10^{-7}M)$. It is suggested that the inhibition of $Na^+,\;K^+-ATPase$ by cholinergic drugs may be considered as one part of mechanism of cholinergic transmission.

• 한국동물학회지
• /
• 제23권2호
• /
• pp.61-68
• /
• 1980

발정주기에 EK라서 생쥐자궁의 alkaline phosphatase와 transport ATPases의 활성변화를 알아보기 위하여 정량적으로 분석하였다. 발정주기의 각 시기에 있어서 이 효소활성들의 비율은 대체로 그 양상이 서로 비슷하나, 발정기의 $K^+$-dependent와 $Na^+, K^+$-activated ATPases를 제외한 다른 효소들의 활성은 다른 어떤 시기보다도 유의하게 (p<0.025) 높았다. 즉, $K^+$-dependent와 $Na^+, K^+$-activated ATPases의 활성은 발정간기에서 발정기에 이르는 동안 무시할 정도이고, 다만 발정후기에 약간의 활성(0.04$\\sim$0.05 $\\mu$M/mg protein/hr), 총활성의 6$\\sim$7%)이 나타났다. 한편, 발정기에서 $Mg^++$-dependent phosphatase, transport ATPase와 alkaline phosphatase의 활성들은 급속히 현저하게 증가하였으며 각각 0.69(35%), 0.42(21%), 1.58(79%)였다. Alkaline phosphatase는 전 발정주기를 통해 0.60$\\sim$1.58(79$\\sim$90%)의 활성을 보여 그 주종을 이루었다. Alkaline phosphatase의 활성중에는 $Mg^++$-dependent의 것이 총활성의 12$\\sim$16%로 추정되었다. 그러므로 $K^+$-dependent와 $Na^+$-activated ATPases는 발정기 때에 자궁액의 누적을 조절하는 요인이 아니고 발정후기에는 자궁상피 속으로 내액을 재흡수하는 요인인 것으로 짐작되며, EH한 $Mg^++$-dependent phosphatase, transport ATPase 그리고 alkaline phosphatase는 생쥐의 자궁 상피세포에서부터 내액을 분지하는 데에 밀접히 관련되어 있는 것으로 사려된다.

• The Korean Journal of Physiology
• /
• 제29권2호
• /
• pp.269-277
• /
• 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.

• 약학회지
• /
• 제30권1호
• /
• pp.31-41
• /
• 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.

• 한국환경농학회지
• /
• 제21권2호
• /
• pp.102-108
• /
• 2002

염류장애에 의한 작물의 생육저해 원인을 밝혀내기 위하여 상추를 대조양액과 대조양액에 30 mM 및 50 mM $KNO_3$를 첨가하여 염류농도를 높인 양액 등 3가지 조건에서 재배하였으며, 이때 이들 양액의 EC는 각각 1.0, 4.5, 6.5 dS/m 이었다. 뿌리세포의 원형질막 및 액포막에 위치한 $H^+$-ATPase 활성은 각각에 특이적 저해제인 vanadate와 $NO_3^-$를 이용하여 측정하였다. 대조양액에서 재배한 상추 뿌리의 ATPase 활성은 $356\pm1.5$ nmol/min/mg protein이었으며, 30 mM과 50 mM $KNO_3$를 첨가한 양액에서는 활성이 대조활성에 비하여 각각 1.6배, 1.9배 증가하였다. 이것은 상추가 염류장애시 뿌리조직의 ATPase 활성증가를 통하여 적응함을 보여주는 것이며, 활성증가는 주로 액포막 $H^+$-ATPase 활성증가에 의해 이루어짐을 확인하였다. 마이크로솜 ATPase 활성에 미치는 여러 가지 중금속 이온들의 효과를 측정하였으며, 중금속 이온은 100 ${\mu}M$ 농도에서 콩류에 따라 활성을 10$\sim$25% 저해하였다. 특히, $Cu^{2+}$는 주로 액포막 $H^+$-ATPase 활성을 저해함을 확인하였다. 본 연구의 결과로부터 상추는 염류집적 환경에서 뿌리의 ATPase 활성, 특히 액포막에 위치한 $H^+$-ATPase 활성을 증가시켜 적응하며, $Cu^{2+}$는 주로 액포막 $H^+$-ATPase 활성을 저해하는 성분으로 뿌리의 ATPase 활성변화 연구에 유용하게 이용될 수 있음을 확인하였다.

• The Korean Journal of Physiology
• /
• 제17권2호
• /
• pp.125-133
• /
• 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.