The interaction of cations with the sodium pump in red blood cells
An investigation has been carried out into the interactions of some cations with the sodium pump of red blood cell membranes. Two mechanisms for this system have been proposed previously: one is a two- step (consecutive) mechanism in which sodium ions are transported in one step and then potassium ions in a separate, second step. An alternative mechanism involves simultaneous attachment and transport of both ions. The experiments presented involve studies of pump activation by either reactant at various concentrations of the other. A requirement for these studies is that products of the reaction be absent. This indicates the use of a cellular system with its inherent asymmetry and the modification of such a system to allow changes in intracellular ionic composition. This has been achieved with human and pig red blood cells modified by a cation loading procedure involving the sulphydryl inhibitor p-chloromercuribenzene sulphonic acid. Pump activity has been measured mainly as the ouabain-sensitive influx of 42K+-labelled potassium and 134Cs+-labelled caesium, which is a congener for potassium. The results presented in the first section of the thesis confirm and extend a previous result showing that the affinity for external potassium or caesium is dependent on the intracellular sodium concentration. The second section of results demonstrates that the reverse trans-membrane influence applies - that is, a dependence of intracellular sodium affinity on the extracellular cation concentration. These results, together with those in a third section which demonstrate an influence of intracellular magnesium levels on external cation binding, are consistent with and explicable in terms of not only simultaneous binding of sodium, potassium or caesium and magnesium to the pump, but also a random order of attachment.