• 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 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.

• 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.

• 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.

• 한국토양비료학회지
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• 제15권4호
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• pp.241-250
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• 1982

발아초기 녹두의 부위별 alkaline inorganic PPase의 활성변화 및 잎 부위에서 부분 정제하여 얻은 효소를 이용하여 효소적 성질을 조사한 결과를 요약하면 다음과 같다. 1. 잎 부위가 타 부위보다 약 2~4배의 더 높은 활성을 나타냈으며 발아가 진행됨에 따라 잎과 뿌리 그리고 지엽에서는 효소 활성이 초기에 증가하다가 점차 감소하는 경향을 보인 반면, 상배축에서는 초기부터 계속 감소하였으며 하배축에서는 계속 증가하는 경향을 보였다. 2. 정제 과정동안 23.9%의 수율로 86배가 정제되었으며 전기 영동상의 Rm value는 0.35였으며 homogenenity는 아니었고, Km value는 0.89mM로 나타났다. 3. 이 효소는 $Mg^{2+}$에 대해 대단히 Specific하였으며 $Cu^{2+}$와 $Fe^{2+}$도 $Mg^{2+}$에 비해 각각 56%, 55%의 activating effect를 나타냈다. 그러나, $Cu^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Co^{2+}$와 $Ni^{2+}$은 이 효소에 대해 저해체로 작용하였다. 4. 이 효소는 pH 8-9와 $50^{\circ}C$에서 최대의 활성을 보였으며 열에 대해서도 상당히 안정하였다.

• Applied Microscopy
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• 제23권2호
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• pp.53-77
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• 1993

In this study, an attempt was made to investigate the probable organelles participating in the secretion of biligrafin. The animals (ICR male mice, 25-30gm) were divided into normal control and 6 biligrafin injected groups to which 30% biligrafin (0.006ml/gm b.w.) were injected at 10, 20, 40, 80, 160 and 320 min prior to the sampling. The mice of each group were perfused through the heart with ice-cold 2.5% glutaraldehyde buffered with 0.1M Na-cacodylate (pH. 7.4) under the Na-pentobarbital (Nembtal 0.0015mg/gm b.w.) anesthesia and liver tissues were taken from each group. Some specimens were immersed 1 hr in the same solution used in the perfusion. After an overnight rinse in 0.1M Na-cacodylate buffer containing 10% DMSO and 7.6% sucrose, $75{\mu}m$ fronzen sections were made for cytochemical study. The sections were incubated in thiamin pyrophosphatase (TPPase) and inosine diphosphatase (ID Pase) media for 70 min at $37^{\circ}C$ respectively and acid phosphatase (AcPase) medium for 40 min at $37^{\circ}C$. They were postfixed in 1 % $OsO_4$ for 1 hr. The other specimens were immersed for 8 hrs in the fixative consisting of 2.5% glutaraldehyde and 3.0% paraformaldehyde buffered with Na-cacodylate (pH. 7.4). All of the osmificated specimens were processed for electron microscopy. In both normal and biligrafin injected groups, endoplasmic reticulum (ER), vacuoles, Golgi apparatus and lysosomes were seen in the vicinity of bile canaliculus. In the biligrafin injected groups, however, the Golgi apparatus appeared to be decreased and ER and vacuoles were dilated and increased. The rough endoplasmic reticulum (RER) having a few attached ribosomes appeared to be the round saccule, especially at 20 min after biligrafin injection. Smooth endoplasmic reticulum (SER) seemed to be formed by the detachment of ribosomes at the cisternal end of RER. The cistern of SER showed saccules which probably budded off to form the vacuole. The vacuoles were devoid of visible centents. This finding seemed to be in agreement with the biochemical property of the bile constituents. The fusion between the vacuoles and bile canaliculus were frequently seen in the groups injected with biligrafin. The lysosome did not show any changes in the biligrafin injected groups. Accumulation of some material and lipid droplets were seen at the 40 and 80 min after biligrafin injection, especially at the latter. At 160 and 320 min after biligrafin injections, however, they were decreased successively while the RER stack, free ribosomes and polysomes were increased. Although the reactive products of TPPase and IDPase were observed in the ER saccules and vesicles of the normal control and biligrafin injected groups, the fusion between the bile canaliculus and saccules or vesicles could easily be seen in the latter. The AcPase activity, however, was observed in the cistern at the maturing face of Golgi apparatus and lysosomes in both normal and biligrafin groups. The results suggest that the biligrafin is excreted via the vesicles, vacuoles or sacoules probably derived from the SER without the participation of Golgi apparatus and lysosomes, and the excess amount of material is stored as inclusions during the repairing of the organelles being overactive.