Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling

Abstract

Inositol polyphosphates, in their water-soluble or lipid-bound forms, represent a large and multifaceted family of signalling molecules. Some inositol polyphosphates are well recognised as defining important signal transduction pathways, as in the case of the calcium release factor Ins(1,4,5)P 3 , generated by receptor activation-induced hydrolysis of the lipid PtdIns(4,5)P 2 by phospholipase C. The birth of inositol polyphosphate research would not have occurred without the use of radioactive phosphate tracers that enabled the discovery of the ''PI response''. Radioactive labels, mainly of phosphorus but also carbon and hydrogen (tritium), have been instrumental in the development of this research field and the establishment of the inositol polyphosphates as one of the most important networks of regulatory molecules present in eukaryotic cells. Advancements in microscopy and mass spectrometry and the development of colorimetric assays have facilitated inositol polyphosphate research, but have not eliminated the need for radioactive experimental approaches. In fact, such experiments have become easier with the cloning of the inositol polyphosphate kinases, enabling the systematic labelling of specific positions of the inositol ring with radioactive phosphate. This approach has been valuable for elucidating their metabolic pathways and identifying specific and novel functions for inositol polyphosphates. For example, the synthesis of radiolabelled inositol pyrophosphates has allowed the discovery of a new protein posttranslational modification. Therefore, radioactive tracers have played and will continue to play an important role in dissecting the many complex aspects of

Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry

“…The metabolic complexity of InsP turnover and their low abundance, in combination with the absence of a chromophore, and high charge density, have hampered research into these signaling molecules. The most widely applied quantitation technology relies on metabolic labeling of cells using tritiated [ 3 H]inositol, followed by acidic extraction, strong anion exchange high performance liquid chromatography (SAX-HPLC), and manual scintillation counting of individual fractions 11 . While this approach is sensitive, it requires a dedicated radioactive suite, is expensive, and labor and time consuming.…”

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

“…The high charge density of PP-InsPs makes their separation difficult, and the occurrence of PP-InsP regioisomers further complicates these efforts. As a result, most experiments relied on quantitation by metabolic radioactive labeling of cells using [ 3 H]-inositol, as background from the matrix is excluded and high sensitivity is achieved 17,18 . However, this method is costly, timeconsuming, and does not allow to properly distinguish related PP-InsP regioisomers.…”

Pho85 and PI(4,5)P2 regulate different lipid metabolic pathways in response to cold

“…Such Plc1 activation could be revealed by an increase in DPIPs levels, as occurs in the inp51 mutant [4]. However, DPIPs are traditionally evaluated by using radioactive experimental approaches [67] that require a highly controlled research environment and sophisticated equipment to extract and purify radioactive derivatives. In addition, the acidic conditions often used for their analysis appear to cause changes in the profile of DPIPs, in particular of IP 7 and IP 8 [68].…”