• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.301-305
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• 2002

The gene (ppaT) encoding Thermus caldophilus GK24 pyrophosphatase (Tca pyrophosphatase) was cloned and sequenced. The gene was found to contain an open reading frame encoding 175 amino acids with a calculated mass of 19,155 Da. The ppaT gene was expressed under the control of the tac promoter in Escherichia coli. The recombinant Tca pyrophosphatase was purified 21.4-fold with $56\%$ yield and specific activity of 25.7 U $mg^-1$, following a combination of heating (to denature the E. coli proteins) and one step of DEAE-Sephacel column chromatography. The native enzyme was found to have an approximate molecular mass of 110,000 Da and consisted of six subunits. The enzyme exhibited maximal activity at pH of 8.0-8.5 and was stable at $80-90^{\circ}C$. A divalent cation was absolutely required for the enzyme activity, with $Mg^2+$. being the most effective.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1144-1148
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• 2006

DNA can oxidatively be deaminated by ROS, which converts DNA base amino groups to keto groups and can trigger abnormal mutations, resulting in mutagenesis in organisms. In this study, a noncanonical purine dNTP pyrophosphatase (AfPPase) from a hyperthermophilic archaeon Archaeoglobus fulgidus, which hydrolyzes aberrant nucleoside triphosphates, was overexpressed in E. coli, purified, and characterized. The purified AfPPase showed remarkably high activity for XTP and dITP, suggesting that the 6-keto group of these nucleotides is critical for the reactivity. Under optimal reaction conditions, the reaction rate for these substrates was about 120 times that with dGTP. Therefore, AfPPase may play a significant role in DNA repair by hydrolysis of noncanonical nucleotides before they are misincorporated into DNA.

• Bulletin of the Korean Chemical Society
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• 제30권10호
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• pp.2485-2488
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• 2009

• Archives of Pharmacal Research
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• 제26권10호
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• pp.826-831
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• 2003

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose (ADPR) into AMP and ribose-5'-phosphate. It is classified into two groups, $Mg^{2+}$-dependent and $Mg^{2+}$-independent ADPRase, depending on its $Mg^{2+}$requirement. Here, we purified $Mg^{2+}$-dependent ADPRase from rabbit liver and examined what factors affect $Mg^{2+}$ requirement. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that ADPRase is a dimer made up of two identical subunits. $Mg^{2+}$-dependent ADPRase with the highest ADPR affinity had a $K_m$ of 160$\pm$10 $\mu$M and a pH optimum of around pH 9.5. Treatment of the purified ADPRase with heated cytosol fractions at 37$^{\circ}C$ for 3 h caused some changes in the chemical properties of the enzyme, including an increase in molecular weight, a decrease in solubility, and a loss of $Mg^{2+}$-dependency. The molecular weight of the cytosol-treated ADPRase measured by gel filtration was over 420 kDa, suggesting, for the first time, that ADPRase could be polymerized by undefined cytoplasmic factors, and that polymerization is accompanied by changes in the solubility and metal ion dependency of the enzyme.

• Journal of Plant Biotechnology
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• 제40권3호
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• pp.156-162
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• 2013

한발에 대한 식물체의 내성은 수분이 부족한 환경에서도 안정적으로 작물의 생산성을 유지하기 위해 필요한 유용한 특성 중 하나이다. 우리는 애기장대의 $H^+$-pyrophosphatase (AVP1)를 발현하도록 형질전환된 배추가 내건성과 관련되어 있는 몇몇 생리적 척도에 있어 향상됨을 검증하였다. 관수중단 처리로 조성된 토양수분 결핍 조건에서 AVP1 발현 식물체는 비형질전환체와 비교하여 비록 외형적 위조의 정도로는 그 차이를 구별할 수 없었지만 형질전환체가 재식된 토양의 수분포텐셜이 비형질전환체 재식 토양에 비해 더 빠르게 낮아졌다. 이는 형질전환체 잎의 상대적 수분함량이 비형질전환체에 비해 더 높은 것과 연관되어 있는 것으로 사료된다. 또한 건조스트레스 환경에서 비형질전환체에 비해 형질전환체는 광계II 양자수율이 높은 반면 전해질누출과 활성산소족의 하나인 $H_2O_2$와 3,3'-diaminobenzidine의 반응산물이 적었다.

• IMMUNE NETWORK
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• 제5권4호
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• pp.199-204
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• 2005

Background: ADP-ribosyl pyrophosphatases (ADPRase) has been known to catalyze the hydrolysis of ADP-ribose to ribose-5-phosphate and AMP. The role of ADPRase has been suggested to sanitize the cell by removing potentially toxic ADP-ribose. In this study, we examined the effect of nitric oxide on ADPRase activity in macrophages. Methods: ADPRase activity was measured in NO-inducing J774 cells. For in vitro experiments, recombinant human ADPRase was prepared in bacteria. Results: ADPRase activity was increased by the treatment of exogenous NO generating reagent, sodium nitroprusside (SNP), in J774 cells. The increased ADPRase activity was mediated by the post-translational modification, likely to cause cADP-ribosylation via nitrosylation of cysteine residue on the enzyme. The stimulation with endogeneous NO inducers, $TNF-{\alpha}/IFN-{\gamma}$, also increased ADPRase activity through NO synthesis. Futhermore, ADPRase activity may be mediated by the post-translational modification of ADPRase, ADP-ribosylation. Conclusion: These results indicate that NO synthesized by macrophage activation plays a critical role in the increase in ADPRase activity following ADP-ribose metabolism.

• Journal of Chest Surgery
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• 제22권1호
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• pp.16-24
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• 1989

To investigate the effects of hydrocortisone on new-born rat cardiac endothelial cells in culture, the endothelial cells were isolated by means of enzyme-cocktail method. The cells were cultivated in Lees modified Dulbeco\ulcorner medium and 10[M or 10[M of hydrocortisone was added to the medium. The cells were harvested or coverglass and processed for thiamin pyrophosphatase reaction and Feulgen reaction. The enzymatic activities of Golgi complex, number of cells and number of large nucleated[more than tetraploid] cells were counted and discussed for their significance. The results were summarized as follows; 1. Hydrocortisone seemed to accelerate the rate of recovery of cardiac endothelial cells from isolation damage. 2. Endothelial cells treated with hydrocortisone revealed strong positive reaction to thiamine pyrophosphatase in early culture and 10 M group had stronger reaction than that of 10 AM group 3. Hydrocortisone had inhibiting effects on endothelial proliferation and the higher the concentration of the reagent was the stronger effects. 4. Hydrocortisone inhibited the appearance of large nucleate cells in endothelial cell population. 5. Hydrocortisone seemed to suppress the nuclear DNA synthesis.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2003년도 한국생물과학협회 학술발표대회
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• pp.162.2-162
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• 2003

No Abstract, See Full Text

• 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.

• Journal of Yeungnam Medical Science
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• 제5권2호
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• pp.111-119
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• 1988

효소와 기계적인 힘을 이용하여 얻어진 심장세포를 심근세포와 내피세포를 분리하여 48시간 배양한 후 양군으로 나눠서 관찰하였다. 실험군은 배양중에 10% DMSO에 1시간 처리하였고 DMSO를 처리하지 않고 계속 배양한 것을 대조군으로 하였다. DMSO효과를 관찰하기 위하여 도립현미경하에서 세포의 형태, 수축능력, 증식 능력등을 관찰하였고, 심근세포에서는 succinate dehydrogenase 반응으로 사립체의 증감을 대조군과 비교하였고, 심장내과세포에서는 thiamine pyrophosphatase의 반응으로 Golgi의 양을 대조군으로 비교하였다. 실험 결과를 요약하연 다음과 같다. 1. DMSO에 의하여 심근세포는 분화된 형태로 모양이 길어졌고 사립체의 증가에 의해 succinate dehydrogenase의 반응이 대조군보마 강하게 나타났다. 2. DMSO에 의해 심근세포는 근세섬유의 파괴와 혼란이 초래되었고 이로 인하여 수축능력이 감소되었다. 3. 심장내과 세포는 DMSO에 의해 세포증식이 감소되고 고유기능이 강조되어 Golgi의 표지효소안 thiamine pyrophosphatase의 반응이 대조군보다 강하였다. 4. 심장내과세포는 DMSO의 세포접착력 소실과 운동성 소실작용에 의해 배양중 많은 세포의 손실이 초래되었다.