Different chain length specificity among three polyphosphate quantification methods Ryo Ohtomo a, * , Yoko Sekiguchi b , Tomoko Kojima a , Masanori Saito c,1 a National Agricultural Research Organization, National Institute of Livestock and Grassland Science, Nasushiobara, Tochigi 329-2793, Japan b Nippon Dionex K.K., Yokogawa-ku, Osaka 532-0011, Japan c National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan article info Article history: Received 20 May 2008 Available online 7 August 2008 Keywords: Polyphosphate Chain length Polyphosphate kinase (PPK) Polyphosphate exopolyphosphatase (PPX) abstract Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbus- cular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which poly- phosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples. Ó 2008 Elsevier Inc. All rights reserved. Inorganic polyphosphate (poly P) 2 is a naturally occurring linear polymer of phosphate (Pi). It is widely distributed in living organ- isms [1,2] and is suggested to have various biological functions such as Pi reserve, energy source, cation sequestration and storage, mem- brane transport, cell envelope formation, and regulations of enzyme activity, translation, and transcription [1,3]. Arbuscular mycorrhizal (AM) fungi, colonizing to plant root and forming the most widespread symbiosis in terrestrial ecosystems to support nutrient (especially phosphorus) acquisition of the host plant, has been known to accumulate poly P in a large amount [4]. The poly P accumulated in extraradical hyphae of the fungi is considered to be important for long-distance translocation of Pi from extraradical to intraradical hyphae [5,6] but not as the main phosphagen for their own metabolism [7]. Within a host plant root, poly P is believed to be degraded into Pi prior to its delivery to the host in exchange for photosynthates from the host [5,8]. Thus, poly P is considered as a critical compound for the key ecological functions of AM fungi. A specific and sensitive poly P quantification method using Escherichia coli poly P kinase (PPK) was developed more than 10 years ago [9]. The method was used to study rapid Pi uptake and poly P synthesis in the extraradical hyphae of AM fungi, Archaeos- pora leptoticha [10]. More recently, Ohtomo and Saito [11] demon- strated that the intensity of colonization and the poly P content were highly correlated in onion root colonized by Gigaspora marga- rita. Because the concentration of poly P was found to be low in nonmycorrhizal plant root [11–13], poly P can be a useful molecu- lar marker to evaluate Pi-supplying activity of AM fungi [11]. The PPK method is an outstanding and powerful tool for poly P analysis because it is highly sensitive and specific, but Ohtomo and coworkers [14] suggested that the method was not applicable to short poly P whose chain length was 20 Pi residues or less. Our preliminary experiment to monitor poly P content of mycorrhizal soybean (Glycine max) root colonized by Glomus sp. R10 detected only a trace amount of poly P by the PPK method (T. Kojima et al., unpublished result). Whether this unexpected result was just an artifact due to low sensitivity of PPK to short-chain poly P or an indication that poly P accumulation is not a common feature of AM fungi cannot be known unless another method covering a wider poly P chain length becomes available. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.08.002 * Corresponding author. Fax: +81 287 36 6629. E-mail address: rotm@affrc.go.jp (R. Ohtomo). 1 Present address: Tohoku University, Osaki, Miyagi 989-6711, Japan. 2 Abbreviations used: poly P, polyphosphate; Pi, phosphate; AM, arbuscular mycorrhizal; PPK, poly P kinase; TBO, toluidine blue O; P 2 , sodium pyrophosphate; P 3 , sodium tripolyphosphate; ATP, adenosine triphosphate; ADP, adenosine diphos- phate; PPX, poly P exopolyphosphatase; IPTG, isopropyl b-d-1-thiogalactopyranoside; BME, b-mercaptoethanol; BPB, bromophenol blue; TBE, Tris–borate–EDTA; EDTA, ethylenediaminetetraacetic acid; PP1, poly P fraction 1; PAGE, polyacrylamide gel electrophoresis; XC, xylen cyanole; IC, ion chromatography; BSA, bovine serum albumin; BCA, bicinchoninic acid; AMP, adenosine monophosphate; NMR, nuclear magnetic resonance. Analytical Biochemistry 383 (2008) 210–216 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio