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

Abstract

In muscle, ATP forms a major cytosolic Mg2+ buffer since almost 90% of the intracellular ATP is bound to Mg2+. Despite the importance of this binding only six values of the apparent dissociation constant (K(app)) have been published under physiological conditions and all are at pH 7.2. Using the recently introduced Mg2+ macroelectrodes, K(app) has been measured at 25 and 37°C from pH 5.35 to 8.5 in an intracellular-like milieu. The results could be fitted assuming binding of Mg2+ to both ATP4- and H-ATP3- which implies that the use of the Beck equation to correct K(app) for pH is inappropriate. This affects 31P-NMR estimations of the intracellular ionized Mg2+ concentration ([Mg2+](i)) which, upon recalculation using the K(app) values in this paper, become similar to measurements using other methods. Since the pK value for ATP protonation is around 6.5, a reduction in the Mg2+ buffering capacity of ATP and a subsequent increase in [Mg2+](i)] would only be expected at pH values less than 6.5. ATP also binds to polyamines. The measured K(app) between spermine and ATP was 662 ± 0.08 μmol l-1 (mean ± S.D., n = 10). Thus, changes in the polyamine concentration by a factor of 3, which could occur in hypertrophy in heart, would cause the release of about 0.7 mmol l-1 Mg2+ from ATP. Adding Mg-ATP2- to an ATP buffer solution causes an increase in the [Mg2+](i). Thus, the proposal of Romani et al. that adrenaline causes release of Mg-ATP2- from mitochondria is incomplete because no change in [Mg2+](i) has been measured. Furthermore, the proposed subsequent loss of Mg2+ from the cell has to be compensated for after adrenaline action to maintain a steady state. Apart from being well buffered in the cytosol, [Mg2+](i) is maintained in the millimolar range by transport systems located in the cell membrane and intracellular organelles. Because of this and because Mg2+ is in the millimolar range, Mg2+, unlike Ca2+, is not suited for cellular transduction over time intervals of seconds or less. Changes in [Mg2+](i) would rather be expected to occur over minutes or longer, making Mg2+ more suited to intracellular modulation rather than transduction.