Abstract
Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4′,6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the μg/ml range. Here, we report that long-wavelength excitation (≥400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.
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Kornberg A, Rao NN, ult-Riche D (1999) Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem 68:89–125
Kulaev IS, Kulakovskaya TV, Andreeva NA, Lichko LP (1999) Metabolism and function of polyphosphates in bacteria and yeast. Prog Mol Subcell Biol 23:27–43
Kumble KD, Kornberg A (1995) Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270:5818–5822
Wang L, Fraley CD, Faridi J, Kornberg A, Roth RA (2003) Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells. Proc Natl Acad Sci U S A 100:11249–11254
Pavlov E, Zakharian E, Bladen C, Diao CT, Grimbly C, Reusch RN, French RJ (2005) A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction. Biophys J 88:2614–2625
Abramov AY, Fraley C, Diao CT, Winkfein R, Colicos MA, Duchen MR, French RJ, Pavlov E (2007) Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death. Proc Natl Acad Sci U S A 104:18091–18096
Kim D, Cavanaugh EJ (2007) Requirement of a soluble intracellular factor for activation of transient receptor potential A1 by pungent chemicals: role of inorganic polyphosphates. J Neurosci 27:6500–6509
Hess HH, Derr JE (1975) Assay of inorganic and organic phosphorus in the 0.1–5 nanomole range. Anal Biochem 63:607–613
Ault-Riche D, Fraley CD, Tzeng CM, Kornberg A (1998) Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. J Bacteriol 180:1841–1847
Rao NN, Liu S, Kornberg A (1998) Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 180:2186–2193
Tijssen JP, Beekes HW, Van SJ (1982) Localization of polyphosphates in Saccharomyces fragilis, as revealed by 4′,6-diamidino-2-phenylindole fluorescence. Biochim Biophys Acta 721:394–398
Zeng RJ, Saunders AM, Yuan Z, Blackall LL, Keller J (2003) Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms. Biotechnol Bioeng 83:140–148
Onda S, Hiraishi A, Matsuo Y, Takii S (2002) Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system. J Gen Appl Microbiol 48:43–54
Pallerla SR, Knebel S, Polen T, Klauth P, Hollender J, Wendisch VF, Schoberth SM (2005) Formation of volutin granules in Corynebacterium glutamicum. FEMS Microbiol Lett 243:133–140
Ruiz FA, Lea CR, Oldfield E, Docampo R (2004) Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem 279:44250–44257
Rodrigues CO, Ruiz FA, Rohloff P, Scott DA, Moreno SN (2002) Characterization of isolated acidocalcisomes from Toxoplasma gondii tachyzoites reveals a novel pool of hydrolyzable polyphosphate. J Biol Chem 277:48650–48656
Liu WT, Nielsen AT, Wu JH, Tsai CS, Matsuo Y, Molin S (2001) In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process. Environ Microbiol 3:110–122
Klauth P, Pallerla SR, Vidaurre D, Ralfs C, Wendisch VF, Schoberth SM (2006) Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum. Appl Microbiol Biotechnol 72:1099–1106
Pavlov E, Grimbly C, Diao CT, French RJ (2005) A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells. FEBS Lett 579:5187–5192
Abramov AY, Scorziello A, Duchen MR (2007) Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation. J Neurosci 27:1129–1138
Wurst H, Kornberg A (1994) A soluble exopolyphosphatase of Saccharomyces cerevisiae. Purification and characterization. J Biol Chem 269:10996–11001
Huang R, Reusch RN (1995) Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations. J Bacteriol 177:486–490
Reusch RN, Sadoff HL (1988) Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes. Proc Natl Acad Sci U S A 85:4176–4180
van Veen HW, Abee T, Kortstee GJ, Pereira H, Konings WN, Zehnder AJ (1994) Generation of a proton motive force by the excretion of metal-phosphate in the polyphosphate-accumulating Acinetobacter johnsonii strain 210A. J Biol Chem 269:29509–29514
Beauvoit B, Rigoulet M, Raffard G, Canioni P, Guerin B (1991) Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply. Biochemistry 30:11212–11220
Akiyama M, Crooke E, Kornberg A (1993) An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268:633–639
Andreeva NA, Okorokov LA (1993) Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope. Yeast 9:127–139
Dunn T, Gable K, Beeler T (1994) Regulation of cellular Ca2+ by yeast vacuoles. J Biol Chem 269:7273–7278
Reusch RN, Huang R, Bramble LL (1995) Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli. Biophys J 69:754–766
Acknowledgements
We grateful to the late Dr. Arthur Kornberg, Department of Biochemistry at Stanford University, for providing us with purified scPPX1 enzyme. This work was supported by operating grants from Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Alberta, NWT, and Nunavut.
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Aschar-Sobbi, R., Abramov, A.Y., Diao, C. et al. High Sensitivity, Quantitative Measurements of Polyphosphate Using a New DAPI-Based Approach. J Fluoresc 18, 859–866 (2008). https://doi.org/10.1007/s10895-008-0315-4
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DOI: https://doi.org/10.1007/s10895-008-0315-4