Methods of Inorganic Polyphosphate (PolyP) Assay in Higher Eukaryotic Cells
Experimental investigation of polyP in higher eukaryotic organisms is a very challenging task. Levels of polyP in higher eukaryotes have several orders of magnitude lower compared to the levels of polyP in bacteria. Although many methods that were develop
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Methods of Inorganic Polyphosphate (PolyP) Assay in Higher Eukaryotic Cells Maria E. Solesio and Evgeny V. Pavlov
Abstract
Experimental investigation of polyP in higher eukaryotic organisms is a very challenging task. Levels of polyP in higher eukaryotes have several orders of magnitude lower compared to the levels of polyP in bacteria. Although many methods that were developed for studies of polyP in bacteria can be adapted for eukaryotes, some important points need to be considered in order to allow investigation of polyP in low abundance situations. In this chapter, we present an overview of the experimental methods of polyP assays in higher eukaryotes that are currently available. These methods include the biochemical approaches of polyP extraction and quantification as well as fluorescent imaging approaches that are suitable for polyP assays in the live tissue culture cells. We discuss advantages and limitations of each of the approaches. Finally, we describe the use of bacterial polyP metabolising enzymes as a tool for studies of polyP in eukaryotic cells.
6.1
Introduction
Inorganic polyphosphate (polyP) assay in biological samples has always been a very challenging experimental problem. The very nature of the polymer makes it difficult to develop a method that will be selective and that will allow to accurately estimate the level of polyP in living organisms. There are two key challenges to the problem:
M.E. Solesio • E.V. Pavlov (*) Department of Basic Sciences, New York University College of Dentistry, 345 East 24th Street, Schwartz Building, Room 1030, New York, NY 10010, USA e-mail: [email protected] © Springer International Publishing Switzerland 2016 T. Kulakovskaya et al. (eds.), Inorganic Polyphosphates in Eukaryotic Cells, DOI 10.1007/978-3-319-41073-9_6
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First, polyP is a polymer made of many orthophosphates linked together by phosphoanhydride bonds in a way similar to ones found in ATP. The reported chain length of polyP polymer is in the range of several phosphate units to several hundreds of phosphate units (Kulaev et al. 2004). Taking into account that polyP conformation is highly variable and that this polymer does not have any specific chemical group in its structure, it is very difficult to develop a probe that can recognise polyP, but at the same time, does not react with any other molecules in the living cell. Second, polyP is highly negatively charged polymer. This makes polyP a highly reactive polymer which can readily interact with divalent ions. Further, polyP has been reported to be able to form stable complexes with other biological polymers like RNA, polyhydroxybutyrate (PHB) and proteins (Kornberg et al. 1999). For this reason, even if a probe that can specifically bind to polyP is used in the assay, the presence of only free polyP in the biological sample or in living cell can be estimated. The pool of polyP that is complexed with other molecules will not be available to the probe. To some extent, the parallel can be made to the use of calci
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