• 대한임상검사과학회지
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• 제41권2호
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• pp.83-92
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• 2009

This study was undertaken to examine the effect of oxidants on membrane transport function in renal epithelial cells. Hydrogen peroxide ($H_2O_2$) was used as a model oxidant and the membrane transport function was evaluated by measuring $Na^+$-dependent phosphate ($Na^+$-Pi) uptake in opossum kidney (OK) cells. $H_2O_2$ inhibited $Na^+$-Pi uptake in a dose-dependent manner. The oxidant also caused loss of cell viability in a dose-dependent fashion. However, the extent of inhibition of the uptake was larger than that in cell viability. $H_2O_2$ inhibited $Na^+$-dependent uptake without any effect on $Na^+$-independent uptake. $H_2O_2$-induced inhibition of $Na^+$-Pi uptake was prevented completely by catalase, dimethylthiourea, and deferoxamine, suggesting involvement of hydroxyl radical generated by an iron-dependent mechanism. In contrast, antioxidants Trolox, N,N'-diphenyl-p-phenylenediamine, and butylated hydroxyanisole did not affect the $H_2O_2$ inhibition. Kinetic analysis indicated that $H_2O_2$ decreased Vmax of $Na^+$-Pi uptake with no change in the Km value. Phosphonoformic acid binding assay did not show any difference between control and $H_2O_2$-treated cells. $H_2O_2$ also did not cause degradation of $Na^+$-Pi transporter protein. Reduction in $Na^+$-Pi uptake by $H_2O_2$ was associated with ATP depletion and direct inhibition of $Na^+$-$K^+$-ATPase activity. These results indicate that the effect of $H_2O_2$ on membrane transport function in OK cells is associated with reduction in functional $Na^+$-pump activity. In addition, the inhibitory effect of $H_2O_2$ was not associated with lipid peroxidation.

• BMB Reports
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• 제29권3호
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• Preventive Nutrition and Food Science
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• 제6권1호
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• pp.43-50
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• 2001

The effects of cardiac ischemia-reperfusion and $\beta$-adrenergic stimulation on metabolic function and energetics were investigated in Lan gendorff-perfused spontaneously hypertensive rat (SHR) hearts. Sarcoplasmic reticulum {TEX}$Ca^{2+}${/TEX}-dependent ATPase and cardiac lactate dehydrogenase (LDH) are additionally studied. The perfusion medium (1.0 mM {TEX}$Ca^{2+}${/TEX}) contained 5 mM glucose(+5 U/L insulin) and 2 mM pyruvate as substrates. Global ischemia was induced by reducing perfusion pressure of 100 to 40 cm {TEX}$H_{2}${/TEX}O, followed by 20 min reperfusin. Isoproterenol (ISO, 1$\mu$M) was infused for 10 min. Coronary vascular resistance and myocardial oxygen consumption ({TEX}$MVO_{2}${/TEX}) of SHR were increased in parallel with enhanced venous lactate during ischemia and reperfusion compared to those of Sprague Dawley (SD) hearts. Although ischemia-induced increase in venous lactate and combined adenosine plus inosine was abolished, coronary vasodilation produced in SD during reperfusion. In SHR, depressed reactive hyperemia was associated with a fall in cardiac ATP and CrP/Pi ratio and a rise in intracellular lactate/Pyruvate ratio. On the other hand, ISO produced coronary functional hyperemia and an increase in {TEX}$MVO_{2}${/TEX}. However, these responses were less than those in SHR hearts. The ATPase activity of SHR was attenuated in free {TEX}$Ca^{2+}${/TEX} concentrations used under basal condition and with ISO compared to that of SD. Venous lactate output and cardiac LDH activity were augmented in SHR as influenced by ISO. These results demonstrate that coronary reactive and functional hyperemia was dpressed in SHR, which cold be explained by alterations in the cytosolic phosphorylation potential and the cytosolic redox state manipulated by LDH, and by abnormal free calcium handling.

• Journal of Radiation Protection and Research
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• 제30권4호
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• pp.197-208
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• 2005

Although little information is available on the underlying mechanisms, various genetic factors have been associated with tissue-specific responses to radiation. In the present study, we explored the possibility whether organ specific gene expression is associated with radiosensitivity using samples from brain, lung and spleen. We examined intrinsic expression pattern of 23 genes in the organs by semi-quantitative RT-PCR method using both male and female C57BL/6 mice. Expression of p53 and p21, well known factors for governing sensitivity to radiation or chemotherapeutic agents, was not different among the organ types. Both higher expression of sialyltransferase, delta7-sterol reductase, leptin receptor splice variant form 12.1, and Cu/Zn superoxide dismutase (SOD) and lower expression of alphaB crystalline were specific for spleen tissue. Expression level of glutathione peroxidase and APO-1 cell surface antigen gene in lung tissue was high, while that of Na, K-ATPase alpha-subunit, Cu/ZnSOD, and cyclin G was low. Brain, radioresistant organ, showed higher expressions of Na, K-ATPase-subunit, cyclin G, and nucleolar protein hNop56 and lower expression of delta7-sterol reductase. The result revealed a potential correlation between gene expression patterns and organ sensitivity, and Identified genes which might be responsible for organ sensitivity.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.351-357
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• 2009

Natural wild-type strains of Bacillus subtilis are extensively used in agriculture as biocontrol agents for plants. This study examined two antagonist B. subtilis strains, KB-1111 and KB-1122, and the results illustrated that KB-1122 was a more potent inhibitor of the indicator pathogen than KB-1111. Thus, to investigate the intrinsic differences between the two antagonist strains under normal culture conditions, samples of KB-1111 and KB-1122 were analyzed using MALDI-TOF-MS. The main differences were related to 20 abundant intracellular and 17 extracellular proteins. When searching the NCBI database, a number of the differentially expressed proteins were identified, including 11 cellular proteins and 10 secretory proteins. Among these proteins, class III stress-response-related ATPase, aconitate hydratase, alpha-amylase precursor, and a secretory protein, endo-l, 4-beta-glucanase, were differentially expressed by the two strains. These results are useful to comprehend the intrinsic differences between the antagonism of KB-1111 and KB-1122.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.673-679
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• 2003

Glutamine and urea, abundant in body fluids or plasma, yield net ammonium ions upon hydrolysis by ${\gamma}-glutamyl$ transpeptidase (${\gamma}-GTP$) and urease, respectively, and these two enzymes are largely produced from Helicobacter pylori. To investigate bacterial potential of ammonium production, we first quantified those in whole-cell systems and found that the relative ratio of their amounts varied greatly, especially with pH values and the cell's aging. During the H. pylori cultivation, the ratio appeared to be inversely proportional to each other, showing a progressive increase of the ${\gamma}-GTP$ with decreasing of the urease. Under the urease-defective conditions due to low pH or coccoids, the bacterial cells still possessed a considerable amount of ${\gamma}-GTP$, which was found exclusively in the external compartment, therefore, the cell's ammonium production was found to be solely dependent upon glutamine, and the external ammonium concentration was constant without any contribution of urea concentration. Such ammonium constancy would definitely have an adverse effect on the host, because of its absolute requirement for vacuolar degeneration by H. pylori VacA, maximized at approximately 10 mM $NH_4Cl$. It was also found that, by using the metal-saturated membrane vesicles, ammonium ions were likely to be involved in the pH-dependent cation-flux across the H. pylori membrane, where the role of ${\gamma}-GTP$ in ammonium homeostasis around cells was suggested, especially under the hostile conditions against H. pylori.

• 약학회지
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• 제30권3호
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• pp.149-156
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• 1986

Many experiments have showed that the sodium and potassium ion transporting system and the Na, $^+K^+$-ATPase activity of membrane fragments are inhibited by digitalis glycosides and that the pump may be associated with the pharmacological receptor for the drugs. The aim of our investigation is to elucidate the ouabain binding sites occupation in heart following infusion of ouabain to intact animals by the $^3H$-ouabain binding assay. Lethal dose and 26 percent of lethal dose of ouabain were infused to intact rabbit through ear vein. Microsomal fraction was fractionated from ouabain treated rabbit heart. $^3H$-ouabain binding to these fraction in vitro was studied by the Schwartz's method. $^3H$-ouabain binding to heart microsomal fraction was also studied following infusion of ginseng ethanol extract and caffeine to rabbits respectively. 1) The infusion of lethal dose ouabain (113$\mu\textrm{g}$/kg) inhibited the specific $^3H$-ouabain binding to rabbit heart microsomal fraction to the level of 60% (p<0.01) of control group and the infusion of 26% of lethal dose of ouabain led to the level of 79% (p<0.01) of the control group. 2) Time course of binding of 0.4$\mu{M}$ $^3H$-ouabain to microsomal fraction from rabbit heart following infusion of lethal and 26% of lethal dose of ouabain showed dose dependence at various incubation time. 3) Compared with control, only slight change of $K_d$ and $B_{max}$ was detected in in vitro $^3H$-ouabain binding after infusion of ginseng ethanol extract (300mg/kg) to rabbit. 4) In caffeine infusion group, $^3H$-ouabain binding yielded nearly the same results as control group.

• 한국식품과학회지
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• 제20권6호
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• pp.862-867
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• 1988

한류성어류(연어, 명태)와 난류성어류(상어)의 열변성과 pH의 상관성에 있어서는 서식하는 환경온도에 관계없이 어류의 근원섬유단백질의 ATPase는 pH $6.5{\sim}8.5$ 범위에서 모두 온도에 의존하는 변성을 나타내었다. 한편 열안정성에 대한 열역학량중 D value와 변성속도 Kd value에 있어서는 상어 >연어 >명태 순으로 확실한 차이를 나타내어 어류 근원섬유단백질의 열안정성은 서식하는 어류의 환경온도와 매우 밀접한 관계가 있는것으로 나타났으며, 동물의 종류에 따라서도 차이를 나타내었다.

• 한국축산식품학회:학술대회논문집
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• 한국축산식품학회 2004년도 정기총회 및 제33차 춘계 학술대회
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• pp.198-201
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• 2004

To investigate hydrostatic pressure (HP) effect on myofibrillar protein (Mf) extracted from bovine Semitendinosus muscle, Ca- and Mg-ATPase activities to evaluate denaturation of myosin and actin, and soluble protein contents were observed. In Mf treated with 100 MPa for 5 min was not observed denaturation of myosin and actin. In Mf treated with 200 MPa for 5 min, denaturation of myosin and actin were observed but inactivation rate was low (0.0136 $min^{-1}$). Inactivation rate of myosin and actin was dramatically increased above 300 MPa treatment. However denaturation of myosin and actin was not that critical with duration time. By increasing pressure size, the amount of myosin and actin in soluble protein eluted in 20 mM potassium phosphate buffer (pH 7.0) containing 0.6 M NaCl were decreased. SDS-PAGE of soluble protein released from Mf suspension in 0.1 M NaCl buffer (pH 7.0) showed that low molecular weight proteins (15${\sim}$36 KDa) were released by HP treatment above 200 MPa. From the results, denaturation of myosin and actin, and release of light molecule proteins of Mf were observed by HP treatment over 200 MPa.

• 한국자기공명학회논문지
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• 제20권1호
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• pp.22-26
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• 2016

FANCJ is a DNA helicase which contributes genome stability by resolving G-quadruplex DNA from 5' to 3' direction. In addition to main ATPase helicase core, FANCJ has the protein binding region at its C-terminal part. BRCA1 and BLM are the binding partner of FANCJ and these protein-protein interactions contribute genomic stability and the proper response to replication stress. As the first attempt for studying FANCJ-BLM interaction, we prepared BLM binding region of FANCJ and characterized with CD and NMR spectroscopy. FANCJ (881-941) with N-ter 6xHis was purified as the oligomer. Secondary structure prediction based on CD data revealed that FANCJ (881-941) composed with ${\beta}$ sheet, turn and coils.$^1H-^{15}N$ HSQC spectra showed nonhomogeneous peak intensities with less number of peaks comparing than the number of amino acids in the construct. It indicated that optimization should be necessary for detailed further structural studies.