• Journal of Microbiology and Biotechnology
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• v.12 no.2
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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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• v.40 no.3
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• pp.156-162
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• 2013

Plant tolerance to drought is a beneficial trait for stabilizing crop productivity under water deficits. Here we report that genetically engineered Chinese cabbage expressing Arabidopsis $H^+$-pyrophosphatase (AVP1) shows enhanced physiological parameters related to drought tolerance. In comparison with wild type plants under soil water deficit stress created by cessation of irrigation, soil water potential in pot with AVP1-expressing plants was more rapidly decreased that might lead to increased relative water content in leaves, while both genotypes had indistinguishable wilting phenotypes. Transgenic plants subjected to drought treatment also exhibited higher photosystem II quantum yield in addition to lower electrolyte leakage and $H_2O_2-3,3^{\prime}$-diaminobenzidine content when compared to wild type plants.

• Archives of Pharmacal Research
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• v.26 no.10
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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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• v.29 no.3
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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.

• Korean Journal of Soil Science and Fertilizer
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• v.15 no.4
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• pp.241-250
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• 1982

Time course of alkaline inorganic PPase activity with different parts of mung-bean sprout and some properties of partially purified enzyme from mung-bean leaves were investigated. The enzyme activity in leaf, root and cotyledon showed a tendency to increase at an early stage and then decrease gradually as germination continued. However, the crude homogenate of epicotyl showed the continuous decline of the enzyme activity but that of hypocotyl showed the continuous increase. In particular, the enzyme activity of leaf fraction was about 2-4 times as high as those of other fractions. The specific activity of the leaf enzyme was increased 86-fold, with a 23.9% yield, upon purification procedures. The purified enzyme from leaves had the Rm value of 0.35 and was not homogenous when judged by disc gel electrophoresis. Using tetrasodium pyrophosphate as a substrate, the apparent Km value for the partially purified enzyme was 0.89 mM. The enzyme was highly specific for $Mg^{2+}$ $CU^{2+}$ and $Fe^{2+}$ was also showed the activating effect of 56% and 55% with $Mg^{2+}$, respectively. However, $Ca^{2+}$), $Zn^{2+}$, $Mn^{2+}$, $Co^{2+}$ and $Ni^{2+}$ acted as inhibitors for the enzyme. The pH optimum for the enzyme shifted from 9.0 to 8.0 as the concentration of $Mg^{2+}$ was increased. The enzyme from mun-bean leaf was the most active at $50^{\circ}C$ and considerably stable on heat.

• Applied Microscopy
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• v.23 no.2
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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.