• 제목/요약/키워드: p-type {H^+}-ATPase$

검색결과 21건 처리시간 0.024초

Cation Flux-Mediated Activation of P-Type ATPase in Helicobacter pylori

  • Yun, Soon-Kyu;Ki, Mi-Ran;Park, Jeong-Kyu;Lim, Wang-Jin;Hwang, Se-Young
    • Journal of Microbiology and Biotechnology
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    • 제10권4호
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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.

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Synergistic Inhibition of Membrane ATPase and Cell Growth of Helicobacter pylori by ATPase Inhibitors

  • Ki, Mi-Ran;Yun, Soon-Kyu;Lim, Wang-Jin;Hong, Bum-Shik;Hwang, Se-Young
    • Journal of Microbiology and Biotechnology
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    • 제9권4호
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    • pp.414-421
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    • 1999
  • Helicobacter pylori were found to be resistant to azide but sensitive to vanadate, suggesting that defect in the P-type ATPase activity rather than F-type ATPase would be lethal to cell survival or growth. To elucidate the relationship between this enzyme inhibition and H. pylori death, we determined the effect of omeprazole (OMP) plus vanadate on enzyme activity and cell growth. The minimum inhibitory concentration (MIC; ca. 0.8$\mu$mol/disk) of vanadate for H. pylori growth was lowered over l0-fold with the aid of OMP, whereby its inhibitory potential toward the P-type ATPase activity was diametrically increased. Alternatively, we found that this enzyme activity was essential for active transport in H. pylori. From these observations, we strongly suggest that the immediate cause of the growth inhibition of H. pylori cells with OMP and/or vanadate might be defective in the cell's active transport due to the lack of P-type ATPase activity. From the spectral data with circular dichroism (CD) spectroscopy, we found that activated OMP (OAS) at concentration below MIC did not disrupt helical structures of membrane proteins. Separately, we determined the cytopathic effect of OAS by SDS-PAGE, indicating the change in the production of cytoplasmic protein but not cell membrane.

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Effect of Omeprazole on Membrane P-Type ATPase and Peptide Transport in Helicobacter pylori

  • KI, MI-RAN;SOON-KYU YUN;SE-YOUNG HWANG
    • Journal of Microbiology and Biotechnology
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    • 제9권3호
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    • pp.235-242
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    • 1999
  • We investigated the growth-inhibitory mechanism of Helicobacter pylori by omeprazole (OMP) and its activated sulfenamide (OAS). Using dithiothreitol (DTT) and 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB; Ellman's reagent), we first determined the relationship between the binding capacity of these compounds to H. pylori membrane and its significance to membrane P-type ATPase activity. After incubation of the intact H. pylori cells with either OMP or OAS, the residual quantity of free SH-groups on the cell membrane was measured, and, the resulting values were plotted as a function of time. From this experiment, we found that there was a considerable difference in the membrane-binding rates between OMP and OAS. At neutral pH, the disulfide bond formation on H. pylori membrane was completed within 2 min of incubation of the intact cells with OAS. By OMP, however, it was gradually formed, exceeding 10 min of incubation for completion, whereby, the extent of P-type ATPase inhibition appeared to be proportional to the disulfide forming rate. From this data, it was suggested that the disulfide formation might directly affect enzyme activity. Since OMP per se cannot yield a disulfide bond with cysteine, it is predicted that the enzyme inactivation must be caused by the OAS form. Accordingly, we postulated that, under the neutral pH, OMP could be converted to OAS in the course of transport. By extrapolating the inhibitory slopes, we could evaluate K₁ values, relating to their minimal inhibitory concentrations (MICs) for H. pylori growth. In these MIC ranges, H. pylori uptake or vesicular export of nutrients such as peptides were totally prohibited, but their effect in Escherichia coli were negligible. From these observations, we strongly suggest that the P-type ATPase activity is essential for the survival of H. pylori cells in particular.

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Characteristics of ATPases Present in Everted Membrane Vesicles of Helicobacter pylori

  • Yun, Soon-Kyu;Hwang, Se-Young
    • Journal of Microbiology and Biotechnology
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    • 제7권3호
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    • pp.167-173
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    • 1997
  • Everted membrane vesicles of Helicobacter pylori were prepared and the membrane-resided ATPases were characterized. For comparison, Escherichia coli membrane ATPases and hog gastric mucosal H,K-ATPase were employed. ATPase assay revealed that the composite enzyme pool was relatively low in specific activities, below 1/10 times than that found in E. coli. According to their inhibitory specificities, most of the ATPase pool appeared to belong to the P-type ATPase, sensitive to vanadate but not to azide. The enzyme pool was extraordinarily resistant against treatment by N,N'-dicyclohexylcarbodiimide (DCCD). Certain monovalent cations, e.g., $K^+$ or $NH_4^{+}$ stimulated the whole enzyme pool only in the presence of $Mg^{2+}$. On the contrary, $Ni^{2+}$ and $Zn^{2+}$ increased enzyme activity rather effectively without the aid of $Mg^{2+}$. Under a defined condition employed, H. pylori cells could retain the membrane ATPase pool to the extent of $17{\%}$ at pH 3.2. Moreover, its activity was most stable in acidic conditions (pH 5.4-6.4). However, cytoplasmic or peripheral ATPase pools were hardly detected under acidity (below pH 4.6).

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토마토 뿌리조직에서 분리한 마이크로솜의 $Ca^{2+}$ 흡수 특성 (Characterization of Microsomal $Ca^{2+}$ Uptake in Tomato Root Tissues)

  • 조광현;김영기
    • Applied Biological Chemistry
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    • 제42권2호
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    • pp.116-122
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    • 1999
  • 식물세포의 세포질 $Ca^{2+}$ 이동과 관련된 $Ca^{2+}$ 수송 특성을 조사하기 위하여, 토마토 뿌리조직으로부터 마이크로솜을 분리하고, $^{45}Ca^{2+}$ 흡수 실험을 수행하였다. 반응용액에 P-type ATPase의 선택적 저해제인 1 mM vanadate와 액포막 $H^+-ATPase$의 선택적 저해제인 50 mM $NO_3^-$를 첨가하였을 때, $^{45}Ca^{2+}$ 흡수는 각각 20%와 33%가 저해되었고, 두 가지 저해제를 동시에 첨가하였을 때, 약 47%가 저해되었다. 이러한 저해효과의 특성을 밝히기 위하여 protonophore인 gramicidin을 처리하여 막을 경계로 형성된 $H^{+}$의 농도기울기를 감소시켰을 때, $^{45}Ca^{2+}$ 흡수는 30% 가량 저해되어, 토마토 뿌리조직 마이크로솜에 $Ca^{2+}/H^+$ antiporter가 존재할 가능성을 확인하였다. Gramicidin의 저해효과는 vanadate에 의한 $^{45}Ca^{2+}$ 흡수 저해 이후에도 대조실험과 같은 정도로 얻어져 vanadate의 저해효과와는 무관한 것이 확인되었다. 그러나, $NO_3^-$를 처리하여 $^{45}Ca^{2+}$ 흡수를 저해시킨 후, gramicidin에 의한 추가저해는 거의 관측되지 않았다. 한편, 동물조직 ER/SR-type $Ca^{2+}-ATPase$의 선택적 저해제인 thapsigargin은 마이크로솜 $^{45}Ca^{2+}$ 흡수를 농도 의존적으로 저해하였으며, $10\;{\mu}M$ 농도에서 최대 저해효과를 나타냈다. Thapsigargin에 의한 저해효과는 $NO_3^-$를 사용하여 액포막 $H^{+}-ATPase$ 활성을 저해하였을 때와 gramicidin을 사용하였을 때, 현저하게 감소하였으며 , vanadate의 효과와는 무관하였다. 이러한 결과는 vanadate가 직접적으로 $Ca^{2+}-ATPase$를 저해하며, $NO_3^-$와 thapsigargin은 액포막에 위치한 $H^{+}-ATPase$의 활성을 저해하여 간접적으로 $Ca^{2+}/H^+$ antiporter를 저해함을 의미한다. 결론적으로, 본 연구의 결과는 토마토 뿌리조직에 $Ca^{2+}$ 이동을 유발하는 주요 효소로서 $Ca^{2+}-ATPase$와 액포막 $Ca^{2+}/H^+$ antiporter가 존재함을 보여준다.

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Altered Regulation of Renal Acid Base Transporters in Response to Ammonium Chloride Loading in Rats

  • Kim, Eun-Young;Choi, Joon-Seok;Lee, Ko-Eun;Kim, Chang-Seong;Bae, Eun-Hui;Ma, Seong-Kwon;Kim, Suhn-Hee;Lee, Jong-Un;Kim, Soo-Wan
    • The Korean Journal of Physiology and Pharmacology
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    • 제16권2호
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    • pp.91-95
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    • 2012
  • The role of the kidney in combating metabolic acidosis has been a subject of considerable interest for many years. The present study was aimed to determine whether there is an altered regulation of renal acid base transporters in acute and chronic acid loading. Male Sprague-Dawley rats were used. Metabolic acidosis was induced by administration of $NH_4Cl$ for 2 days (acute) and for 7days (chronic). The serum and urinary pH and bicarbonate were measured. The protein expression of renal acid base transporters [type 3 $Na^+/H^+$ exchanger (NHE3), type 1 $Na^+/{HCO_3}^-$ cotransporter (NBC1), Na-$K^+$ ATPase, $H^+$-ATPase, anion exchanger-1 (AE-1)] was measured by semiquantitative immunoblotting. Serum bicarbonate and pH were decreased in acute acid loading rats compared with controls. Accordingly, urinary pH decreased. The protein expression of NHE3, $H^+$-ATPase, AE-1 and NBC1 was not changed. In chronic acid loading rats, serum bicarbonate and pH were not changed, while urinary pH was decreased compared with controls. The protein expression of NHE3, $H^+$-ATPase was increased in the renal cortex of chronic acid loading rats. These results suggest that unaltered expression of acid transporters combined with acute acid loading may contribute to the development of acidosis. The subsequent increased expression of NHE3, $H^+$-ATPase in the kidney may play a role in promoting acid excretion in the later stage of acid loading, which counteract the development of metabolic acidosis.

Significance of Urease Distribution across Helicobacter pylori Membrane

  • Gang, Jin-Gu;Yun, Soon-Kyu;Choi, Kyung-Min;Lim, Wang-Jin;Park, Jeong-Kyu;Hwang, Se-Young
    • Journal of Microbiology and Biotechnology
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    • 제11권2호
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    • pp.317-325
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    • 2001
  • For heuristic purposes, the relative ratio of urease contents inside and outside cells was surveyed using nine ureB+ strains of Helicobacter pylori. the ratio of the enzyme specific activity appeared to vary greatly between the various H. pylori strains, ranging from 0.5 to 2.5. Besides the above compartment, urease was also richly found in the membrane fraction, especially in either peripheral or integral form. The urease distribution across the H. pylori membrane was significantly influenced by the ambient pH; the specific activity of external urease was highest at pH 5.5 with a narrow plateau, whereas the internal specific activity was highest within a pH range of 4.5 to 6.5 with a broad plateau. These finding strongly suggest that H. pylori urease is secretory and responded to the external pH. However, at pH 4.0 or below, no urease activity was detected in either the internal or external compartment, although an increase in the color development with 2,4,6-trinitrobenzene sulfonate (TNBS) was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these phenomena may be related to a specific proteolysis in certain proteins, including urease or ${\gamma}$-glutamyl transpeptidase. Interestingly, the effect of ammonium ions n alleviating the enzyme inactivation inside the H. pylori cells was remarkably similar to that of D-glucose. In addition, it would appear that the cation acted as a surrogate of not only $Na^+$ but also $K^+$ thereby increasing the H. pylori P-type ATPase activity. This is of great interest, as it implies that the urease action in H. pylori is indispensible at any locus.

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Adipic acid 저항성 변이주 Leuconostoc paramesenteroides의 내산성 특성 (Properties of Acid Tolerance of the Adipic acid-resistant Mutant of Leuconostoc paramesenteroides)

  • 이중근;이홍석;김영찬;주현규;이시경;강상모
    • 한국식품과학회지
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    • 제32권2호
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    • pp.431-438
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    • 2000
  • Adipic acid에 대해 저항성을 갖도록 변이된 Leuconostoc paramesenteroides (ANaP100) 균주의 내산성 특성을 조사하기 위하여 수소이온 투과도, $H^+-ATPase$ 활성, $Mg^{++}$ 해리도, 원형질막의 지방산 조성을 지표로 삼아 야생균주(LPw)와 비교하였다. 수소이온 투과도의 경우 pH 5에서 $t_{1/2}$ 값이 LPw는 4.3분, ANaP100은 4.8분으로서 변이균주가 다소 내산성이 높았으며 $H^+-ATPase$ 활성은 maximal activity가 Leu. paramesenteroides는 모두 pH 6.0에서 가장 높았고 LPw는 0.59 unit/mg protein, 변이균주는 0.63 unit/mg protein으로서 ANaP100이 LPw보다 활성이 높았다. 세포막의 산 손상(acid damage)에 의한 $Mg^{++}$ 해리도에서도 pH 4.0에서 2시간 경과 후 LPw는 52.2%, ANaP100은 27.3%로서 LPw에 비해 약 1/2가량 $Mg^{++}$이 적게 유출되어 산에 의한 세포막의 손상이 적었다. 원형질막의 지방산 조성은 ANaP100가 $C_{18:1}$은 감소하고, $C_{19:0,\;cyclo}$는 증가하여 내산성이 증대되었으며 또한 adipic acid 첨가시에도 LPw에 비해 우수한 증식을 보였다. 따라서 ANaP100이 LPw에 비해 내산성이 증가되었으며 adipic acid 저항성도 함유하는 것이 확인되었다.

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Adipic acid-resistant 변이주 Leuconostoc mesenteroides의 내산성 특성 (Characteristics of Acid Tolerance of Adipic Acid-Resistant Mutant Strain, Leuconostoc mesenteroides)

  • 이중근;이홍석;김영찬;주현규;이시경;정대현;강상모
    • 한국미생물·생명공학회지
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    • 제28권2호
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    • pp.63-70
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    • 2000
  • To determine an increased acid tolerance of an adipic acid-resistant mutant Leuconostoc mesenteroides(ANaM100) developed for use as a Kimchi starter, proton permeability of cytoplasm, activities of H+-ATPase, Mg++ release and fatty acid composition of cytoplasmic membranes of strain ANaM100 were studied and compared with those of its wild type (LMw). The value of protons permeability of LMw after an acid shock at pH 5.0 was 5.4 min., while the value of ANaM100 cells was 8.4 min. at the same pH. The pH of maximal specific activ-ities of ATPase originated from the LMw and ANaM100 were 0.87 unit/mg protein at pH 6.0 and 0.92 unit/mg pro-tein at pH 5.5, respectively. The release of magnesium ion from ANaM100 was observed about 12.8% at pH 4 after 2 hours, while the wild strains of LMw released Mg++ about 27.6% under the same conditions. The content of C19:0,cyclo and C18:1 in a membrane fatty acid of ANaM100 was higher and lower, respectively than that of LMw. These results indicated that acid tolerance of adipic acid-resistant strain, ANaM100 was significantly improved in comparison with that of its wild type, LMw. In addition, the strain ANaM100 was adipic resistance based on the result of growth of the strain in comparison with that of strain LMw in a broth containing adipic acid.

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Direct Evidence of Intracellular Alkalinization in Saccharomyces cerevisiae KNU5377 Exposed to Inorganic Sulfuric Acid

  • Yun, Hae-Sun;Paik, Sang-Kyoo;Kim, Il-Sup;Jin, Ing-Nyol;Sohn, Ho-Yong
    • Journal of Microbiology and Biotechnology
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    • 제14권2호
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    • pp.243-249
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    • 2004
  • The toxicity of inorganic sulfuric acid as a stressor was characterized in Saccharomyces cerevisiae KNU5377. In this work, we examined physiological responses to low extracellular pH $(pH_{ex})$ caused by inorganic $H_2SO_4$, which could not affect cell growth after pH was adjusted to an optimum with Trizma base. The major toxicity of sulfuric and was found to be reduction of environmental pH, resulting in stimulation of plasma membrane ${H^+}-ATPase$, which in turn contributed to intracellular alkalinization. Using a pH-dependent fluorescence probe, 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (carboxy SNARF-1 AM acetate), to determine $pH_{in}$, we found that color was dependent on the changes of intracellular pH which coincided with calculated $pH_{in}$ of alkalinization up to approximately pH 7.3. This alkalinization did not seem to affect survival of these cells exposed to 30 mM sulfuric acid, which lowered the $pH_{ex}$ of the glucose containing growth media up to approximately pH 3.0; however, the cells could grow only up to 70% of the maximum growth in the same media, when 30 mM sulfuric acid was added.