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1.
A new method for the rapid analysis of inorganic pyrophosphate (PPi) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PPi and is not affected by inorganic phosphate. ATP and PPi can both be analyzed using this method. 相似文献
2.
《BBA》1986,851(2):276-282
Photosynthetic formation of inorganic pyrophosphate (PPi) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PPi synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PPi synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PPi synthesis much less. Even at low rates of electron transport a significant rate of PPi synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPi synthesis is inhibited. It is shown that PPi synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions. 相似文献
3.
A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PPi) was developed. PPi was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl2 and KF. The separated PPi was dissolved by immersing the filter in 0.5 n H2SO4. Inorganic phosphate (Pi) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PPi was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PPi can be accurately assayed even when there is a 104-fold excess of Pi. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PPi. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PPi in the same sample. 相似文献
4.
An enzyme from Entamoeba histolytica catalyzes the formation of acetyl phosphate and orthophosphate from acetate and inorganic pyrophosphate (PPi), but it displays much greater activity in the direction of acetate formation. It has been purified 40-fold and separated from interfering enzyme activities by chromatography. Its reaction products have been quantitatively established. ATP cannot replace PPi as phosphoryl donor in the direction of acetyl phosphate formation nor will any common nucleoside diphosphate replace orthophosphate as phosphoryl acceptor in the direction of acetate formation. The trivial name proposed for the new enzyme is acetate kinase (PPi). 相似文献
5.
The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.Abbreviations PFP
pyrophosphate-fructose-6-phosphate 1-phosphotransferase
- Pi
phosphate
- PPi
pyrophosphate 相似文献
6.
The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of32PPi contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PPi.Enzymes
tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1) 相似文献
7.
A microprocedure for the colorimetric determination of inorganic pyrophosphate (PPi) in the presence or absence of orthophosphate (Pi) has been developed. PPi is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. Pi is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PPi can be detemined. The extinction coefficients are 2.70 × 104 and 8.76 × 103 for PPi and Pi, respectively, both with thiol reagent present, and 2.77 × 103 for Pi with no thiol reagent.A ten-fold excess of Pi does not interfere with the determination of PPi and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PPi estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PPi formation or the rate of synthesis of labeled RNA were very similar. Product PPi formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PPi down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr. 相似文献
8.
In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PPi) in three phototrophic bacteria. Formation of PPi was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PPi synthesis in Rps. viridis was 0.15 mol PPi formed/(min*mol Bacteriochlorophyll) at 23°C. The synthesis of PPi was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PPi synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PPi after short light flashes was also studied. The yield of PPi after a single 1 ms flash was equivalent to approximately 1 mol PPi/500 mol Bacteriochlorophyll. In Rps. viridis chromatophores, PPi was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.Abbreviations BChl
Bacteriochlorophyll
- F0F1-ATPase
Membrane bound proton translocating ATP synthase
- FCCP
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone
- H+-PPase
Membrane bound proton translocating PPi synthase
- TPP+
Tetraphenyl phosphonium ion
- TPB-
Tetraphenyl boron ion
-
Transmembrane electrical potential difference 相似文献
9.
Melina Galizzi Juan M. Bustamante Jianmin Fang Kildare Miranda Lia C. Soares Medeiros Rick L. Tarleton Roberto Docampo 《Molecular microbiology》2013,90(4):699-715
Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PPi, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection. 相似文献
10.
Yoko Chiba ) Ryoma Kamikawa ) Kumiko Nakada-Tsukui ) Yumiko Saito-Nakano ) Tomoyoshi Nozaki ) 《The Journal of biological chemistry》2015,290(39):23960-23970
Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. 相似文献
11.
Shoji Shirota Shiro Watanabe Isao Takeda 《Bioscience, biotechnology, and biochemistry》2013,77(3):325-332
It was found that CDP-choline was formed with good yield from 5′-CMP and choline phosphate or choline chloride by yeast cells. The effects of pyrophosphate (PPi) on the formation of UDPG, GDPM and CDP-choline from respective nucleoside monophosphate by yeast cells were studied. By the addition of PPi to the reaction mixture, the phosphorylation of G-6-P from glucose was inhibited and then the phosphorylation of nucleoside monophosphates was restrained. Such inhibition was reversed by the decomposition of PPi by inorganic pyrophosphatase of yeast cells. The addition of PPi after the formation of nucleotide derivatives caused the accumulation of UTP and GTP and molar yields from nucleotide as substrate was about 80%. But that of CTP was a little in the reaction system of CDP-choline synthesis. Further, this method seems to be suitable for the accumulation of sugar-1-phosphates. 相似文献
12.
Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PPi) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (Pi) concentrations after degradation by inorganic pyrophosphatase of the PPi released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A4, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PPi exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover. 相似文献
13.
Novel bioluminescent quantitative detection of nucleic acid amplification in real-time 总被引:1,自引:0,他引:1
Gandelman OA Church VL Moore CA Kiddle G Carne CA Parmar S Jalal H Tisi LC Murray JA 《PloS one》2010,5(11):e14155
Background
The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories.Principal Findings
Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required.Conclusions
The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. 相似文献14.
This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA
ethylenediaminetetraacetic acid
- PFK(PPi)
pyrophosphate fructose 6-phosphate 1-phosphotransferase
- PPi
inorganic pyrophosphate
-
PPRibP
phosphoribosyl pyrophosphate 相似文献
15.
To elucidate the inhibition mechanisms of hydroxyapatite (HA), a biological model mimicking the mineralization process was developed. The addition of 4% (v/v) dimethyl sulfoxide (DMSO) in synthetic cartilage lymph (SCL) medium containing 2 mM calcium and 3.42 mM inorganic phosphate (Pi) at pH 7.6 and 37 °C produced HA as matrix vesicles (MVs) under physiological conditions. Such a model has the advantage of monitoring the HA nucleation process without interfering with other processes at the cellular or enzymatic level. Turbidity measurements allowed us to follow the process of nucleation, whereas infrared spectra and X-ray diffraction permitted us to identify HA. Mineral formation induced by DMSO and by MVs in the SCL medium produced crystalline HA in a similar manner. The nucleation model served to evaluate the inhibition effects of ATP, GTP, UTP, ADP, ADP-ribose, AMP, and pyrophosphate (PPi). Here 10 μM PPi, 100 μM nucleotide triphosphates (ATP, GTP, UTP), and 1 mM ADP inhibited HA formation directly, whereas 1 mM ADP-ribose and 1 mM AMP did not. This confirmed that the PPi group is a potent inhibitor of HA formation. Increasing the PPi concentration from 100 μM to 1 mM induced calcium pyrophosphate dihydrate. We propose that DMSO-induced HA formation could serve to screen putative inhibitors of mineral formation. 相似文献
16.
Concepción José-Nuñez Alfredo Torres-Larios Leticia Ramírez-Silva Guillermo Mendoza Guillermo Salcedo Armando Gómez-Puyou Marietta Tuena de Gómez-Puyou 《Journal of bioenergetics and biomembranes》2008,40(6):561-568
Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied,
the results have not conclusively shown that the subunits are catalytically active. Since soluble F1 of mitochondrial H+-ATPase can bind inorganic pyrophosphate (PPi) and synthesize PPi from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PPi. The difference spectra in the near UV CD of β-subunits with and without PPi show that PPi binds to the subunits. Other studies show that β-subunits hydrolyze [32P] PPi through a Mg2+-dependent process with an optimal pH of 8.3. Free Mg2+ is required for maximal hydrolytic rates. The Km for PPi is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the
Km for PPi. Thus, isolated β-subunits are catalytically competent with PPi as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to
an increase in catalytic rates. 相似文献
17.
A simple method for measuring PPi at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PPi. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PPi in urine is demonstrated. 相似文献
18.
An enzymatic assay for l-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PPi) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PPi detection system was embodied as three different forms, which allowed PPi to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. l-Methionine could be accurately determined by coupling the PPi detection system and AdoMetS. This AdoMetS coupling assay was highly selective to l-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for l-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in l-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of l-methionine in various kinds of analytes. 相似文献
19.
The levels of phosphorylated compounds studied during the dark period of Crassulacean acid metabolism (CAM) in Kalanchoë leaves showed increases for ATP and pyrophosphate and decreases for ADP, AMP and phosphenolpyruvate; levels of inorganic phosphate remained constant. Changes in adenylate levels and the correlated nocturnal increase in adenylate-energycharge were closely related to changes in malate levels. The increase in ATP levels was much inhibited in CO2-free air and stimulated after induction of CAM in short-day-treated plants of K. blossfeldiana cv. Tom Thumb. Changes in levels of phosphoenolpyruvate and pyrophosphate were independent of the presence of CO2. The results show the operation of complex regulatory mechanisms in the energy metabolism of CAM plants during nocturnal malic-acid accumulation.Abbreviations CAM
Crassulacean acid metabolism
- FW
fresh weight
- OAA
oxaloacetic acia
- PEP
phosphoenol pyruvate
- PPi
pyrophosphate 相似文献
20.
The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5′-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines. 相似文献