John A.S. McGuigan

Mg‐Atp Binding: Its Modification by Spermine, the Relevance to Cytosolic Mg2+ Buffering, Changes in the Intracellular Ionized Mg2+ Concentration and the Estimation of Mg2+ by 31P‐NMR

It is now generally accepted that the intracellular ionized magnesium concentration ([Mg2+]i) in muscle cells is around 1 mmol l−1; in heart muscle this means that from the total some 90‐95% is bound (see McGuigan et al. 1991a). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg‐ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg2+]i using 31P‐NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg2+ and ATP in the cytosol, the [Mg2+]i can be calculated (Vink, 1993).

Use of Ion Selective Microelectrodes to Measure Intracellular Free Mg2

Mg2+ has been dubbed “the forgotten cation” (Brady et al. 1987) for although it is the second most common intracellular cation, the study of its homeostasis and its regulatory function in health and disease has long been neglected. Gradually, however, it is becoming clear that Mg2+ plays an important intracellular role: It is a co-factor in numerous enzymatic reactions, has a regulatory function on several ionic channels, influences Ca2+ uptake into the mitochondria and K+ uptake by skeletal muscle, and may play a crucial role in cardiovascular disease (see reviews by Altura 1988; Elin 1988; Shils 1988; Lauter 1989; White and Hartzeil 1989; Review in Am J Med 1987).

An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions.

In Ca(2+)/Mg(2+) buffers the calculated ionised concentrations ([X(2+)]) can vary by up to a factor of seven. Since there are no defined standards it is impossible to check calculated [X(2+)], making measurement essential. The ligand optimisation method (LOM) is an accurate method to measure [X(2+)] in Ca(2+)/Mg(2+) buffers; independent estimation of ligand purity extends the method to pK(/) < 4. To simplify calculation, Excel programs ALE and AEC were compiled for LOM and its extension. This paper demonstrates that the slope of the electrode in the pX range 2.000-3.301 deviates from Nernstian behaviour as it depends on the value of the lumped interference, Σ. ALE was modified to include this effect; this modified program SALE, and the programs ALE and AEC were used on simulated data for Ca(2+)-EGTA and Mg(2+)-ATP buffers, to calculate electrode and buffer characteristics as a function of Σ. Ca(2+)-electrodes have a Σ < 10(-6) mol/l and there was no difference amongst the three methods. The Σ for Mg(2+)-electrodes lies between 10(-5) and 1.5 (∗) 10(-5) mol/l and calculated [Mg(2+)] with ALE were around 3% less than the true value. SALE and AEC correctly predicted [Mg(2+)]. SALE was used to recalculate K(/) and pK(/) on measured data for Ca(2+)-EGTA and Mg(2+)-EDTA buffers. These results demonstrated that it is pK(/) that is normally distributed. Until defined standards are available, [X(2+)] in Ca(2+)/Mg(2+) buffers have to be measured. The most appropriate method is to use Ca(2+)/Mg(2) electrodes combined with the Excel programs SALE or AEC.

Ionised concentrations in calcium and magnesium buffers: Standards and precise measurement are mandatory

In Ca2+ and Mg2+ buffer solutions the ionised concentrations ([X2+]) are either calculated or measured. Calculated values vary by up to a factor of seven due to the following four problems: The calculated [X2+] in buffers are so inconsistent that calculation is not an option. Until standards are available, the [X2+] in the buffers must be measured. The Ligand Optimisation Method is an accurate and independently verified method of doing this (McGuigan & Stumpff, Anal. Biochem. 436, 29, 2013). Lack of standards means it is not possible to compare the published [Ca2+] in the nmolar range, and the apparent constant (K/) values for Ca2+ and Mg2+ binding to intracellular ligands amongst different laboratories. Standardisation of Ca2+/Mg2+ buffers is now essential. The parameters to achieve this are proposed.