The interaction of magnesium with the sodium pump.

The thesis describes an investigation into the interaction of magnesium with the sodium pump. Three experimental approaches have been made for this study. In the first part, the influence of magnesium on hydrolysis of ATP and pNPP was investigated with plasma membranes from ox brain prepared by a novel method. The preparations had high obtained sensitivities (about 97%) and high specific activities, proving ideal for kinetic studies. It was shown that (Na + K)-ATPase differed strikingly in its response to magnesium and ATP compared to the response of the associated (K)-pNPPase to magnesium and pNPP. Excess magnesium was without effect on pNPPase but inhibited ATPase. Furthermore, Pi inhibited ATP hydrolysis but only at ATP levels suboptimal for activity. The second part deals with the influence of magnesium on protein phosphorylation and the subsequent decomposition of the phosphoprotein. The significant findings were firstly that up to a one hundred-fold excess of magnesium over ATP did not inhibit phosphorylation and secondly that there was no difference in the sensitivity of the phosphoprotein to decay stimulated by ADP or potassium that was dependent upon the level of magnesium present during phosphorylation. In the third part, human red blood cells were used to study the influence of magnesium on active ion movements. Using PCMBS to load red cells with magnesium, it was found that raising internal magnesium inhibited active potassium influx and sodium efflux with little effect on obtain-insensitive fluxes. Moreover, the inhibition of ion movements paralleled inhibition of fragmented membrane ATPase activity. The results provide no support for the mechanism of ATP hydrolysis involving two phosphorylated intermediates and a one step mechanism is preferred. The main finding of physiological significance was that inhibition by magnesium was only evident when pump sites for both sodium and potassium were filled, thereby pointing to a possible regulatory role for internal magnesium.