Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes |
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Authors: | Gosset Gaëlle Satre Michel Blaive Bruno Clément Jean-Louis Martin Jean-Baptiste Culcasi Marcel Pietri Sylvia |
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Affiliation: | aGroupe Sondes Moléculaires en Biologie, Laboratoire Chimie Provence–UMR 6264, Universités d’Aix-Marseille–CNRS, F-13397 Marseille Cedex 20, France;bLaboratoire de Biochimie et Biophysique des Systèmes Intégrés iRTSV/BBSI (UMR 5092 CNRS CEA and Université Joseph Fourier)–CEA, F-38054 Grenoble Cedex 9, France;cCNRS, SYMBIO, Universités d’Aix-Marseille, F-13397 Marseille Cedex 20, France |
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Abstract: | The 31P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pKa, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pKa < 7 allowed an easy and simultaneous 31P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum. |
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Keywords: | 31P NMR α -Aminophosphonates Dictyostelium discoideum Endolysosomal compartment pH determination Exchange broadening |
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