Solution behaviour of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions
Introduction
myo-Inositol hexakisphosphate or phytate (InsP6; its different protonated forms are denoted in this work by H12L, H11L−, …, L12−) is an ubiquitous and abundant molecule in eukaryotic cells (reviewed in [1], [2], [3]). InsP6 can act as a precursor of diphosphoinositol pentakisphosphates (PPInsP5) and bis(diphospho)inositol tetrakisphosphates ((PP)2InsP4) (reviewed in [4]). The rate of metabolic turnover between InsP6 and the derived pyrophosphates is very high [5], [6], [7], especially when considered in relation to the lower concentrations of the latter. It is thus easy to envisage the pyrophosphates as molecular switches regulating cellular processes through phosphorylation–dephosphorylation cycles, and indeed there is evidence some may regulate endocytosis [8], [9] and chemotaxis [7]. In contrast, it has not been so easy to class the biological functions of InsP6 (thought not to be restricted to its role as precursor for the pyrophosphates) to broadly defined categories.
InsP6 interacts strongly with divalent and trivalent cations in a complex, but poorly described, chemistry encompassing solution complexation and precipitation reactions. The lack of a rigorous description of these reactions is a significant hurdle for InsP6 biology. First, it has been a widespread cause of artefactual results in in vitro experiments involving the compound. As pointed out by Shears [1], depletion of Ca2+ and Mg2+ through complexation is one problem. Another problem is unnoticed precipitation (when working with small volumes): this can both deplete the solution of the cation, and generate particulate species that interfere with experimental parameters. Second, it is difficult to extrapolate to the state of InsP6 in vivo, since the physicochemical form of InsP6 in cells is unknown. Most or all of the cellular InsP6 is located in the cytosolic and nuclear compartments either free or bound to cellular components including membranes (see below), and requires chelating agents for its extraction [10]. If it is in free solution it is not entirely clear how InsP6 can exist as such under cytosolic conditions [2]. Shears has suggested that InsP6 may be “wallpapering” the cytosolic face of cell membranes [11], possibly through cation-mediated interactions [12]. Whether soluble or bound to fixed components, it is expected that nuclear and cytosolic InsP6 exist as complexes with Mg(II) and/or Ca(II), but such species have not been defined. InsP6 is not confined to the nuclear/cytosolic compartments in all eukaryotes [13], [14], an observation that extends the possibilities of interactions with Ca(II) in particular. Complexation of Fe(III) by InsP6 has received particular attention, because it is surprisingly effective at preventing the participation of iron in the redox cycles catalysing the formation of the hydroxyl radical [15], [16], [17]. Whether InsP6 can interact with Fe(III) in cells will depend on the relative affinities for Fe(III) and the much more abundant Mg(II).
Qualitative or semi-quantitative data on the InsP6-multivalent cations chemistry have been previously reported (e.g., [18] and references therein, and [19]). The quantitative studies of Bebot-Brigaud et al. [20] and Vasca et al. [21], encompassed a range of divalent cations, but excluded Ca(II) and Mg(II), and employed conditions of temperature and ionic strength deviating from the physiologically most relevant. Two main conclusions stem from these two reports. The first is that, as mentioned, InsP6-metal ion systems comprise two different aspects, namely solution complexation and solid formation. In the presence of excess InsP6, formation of soluble complexes displaying 1:1 stoichiometries predominates. In contrast, when the metal ion is the component in excess, insoluble solids are formed, the compositions of which are mostly unknown. The second conclusion is that the stability constants of the soluble species are strongly influenced by ionic strength, apparently as a result of the formation of stable complexes with the small monovalent cations.
A complete description of the chemistry of InsP6 with multivalent cations would constitute a significant tool for InsP6 biology. We have begun such a study, the initial part of which is reported in this paper. This part comprises the potentiometric determination of the stability constants, at 37.0 °C and I = 0.15 M NaClO4, of the complexes formed by InsP6 and several divalent cations (Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn, Cd), as well as Fe(III) and Al(III). It also comprises the determination of the stability constants for complexes formed between InsP6 and Na(I) at 37.0 °C and I = 0.15 M (Me4N)Cl. In addition, the work includes the isolation and characterization of an unexpected species ([Mg5(H2L)]), predicted to hold particular biological relevance. Finally, the work comprises the study by cyclic voltammetry of the redox exchange between InsP6–Fe(III) and InsP6–Fe(II). These data are of value in themselves as a part of our understanding of InsP6-cation interaction, but more importantly they have allowed us to predict the degrees of association of InsP6 with Mg(II) and Ca(II) in the cytosol of cells, and make inferences on the possibility of its interaction with Fe(III).
Section snippets
Chemicals
All common laboratory chemicals were reagent grade, purchased from commercial sources and used without further purification. CaCl2 · 2H2O, MgCl2 · 6H2O, MnCl2 · 4H2O, Ni(ClO4)2 · 6H2O, ZnCl2, CdCl2, Co(ClO4)2 · 6H2O, CuSO4 · 5H2O, (NH4)2Fe(SO4)2 · 6H2O, Fe(ClO4)3 · xH2O, and Al(NO3)3 were used as metal sources. Solutions of the metals were standardized according to standard techniques [22], [23], [24]. Phytate solutions were prepared by weighing the sodium salt (Sigma), which was verified by elemental analysis
InsP6 protonation equilibria
Values for the protonation equilibrium constants were required in order to study the metal complexation reactions. These protonation constants are known to be strongly influenced by the nature and concentration of the supporting electrolyte [20], [30], [31], [32], [33]. We chose to determine the protonation constants de novo, and to do so both in 0.15 M Me4NCl and in 0.15 M NaClO4. The former, non-interacting, electrolyte would allow determination of protonation constants free from InsP6
Acknowledgements
The authors are grateful to Jorge Castiglioni (LAFIDESU, Facultad de Quimica, Universidad de la República, Uruguay) for thermogravimetric analysis of magnesium phytate, which was carried out on an instrument generously donated by the Japanese Government. This work was supported by DINACYT (Ministry of Education, Uruguay) through a PDT Grant to C.K., PEDECIBA (Uruguay), and Conserjería de Educación, Cultura y Deportes (Canary Islands Government, Spain; grant PI2002/057). R.F.I. is supported by
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