Divalent Cation Interactions with Na,K-ATPase Cytoplasmic Cation Sites: Implications for the para-Nitrophenyl Phosphatase Reaction Mechanism

Abstract

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr(2+) and Ba(2+) did not compete with K(+) for ATPase (an extracellular K(+) effect). Sr(2+) and Ba(2+) did compete with Na(+) for ATPase (an intracellular Na(+) effect) and with K(+) for pNPPase (an intracellular K(+) effect). These results suggest that Ba(2+) or Sr(2+) can bind to the intracellular transport site, yet neither Ba(2+) nor Sr(2+) was able to activate pNPPase activity; we confirmed that Ca(2+) and Mn(2+) did activate. As another measure of cation binding, we observed that Ca(2+) and Mn(2+), but not Ba(2+), decreased the fluorescence of the FITC-labeled pump; we confirmed that K(+) substantially decreased the fluorescence. Interestingly, Ba(2+) did shift the K(+) dose-response curve. Ethane diamine inhibited Mn(2+) stimulation of pNPPase (as well as K(+) and Mg(2+) stimulation) but did not shift the 50% inhibitory concentration (IC(50)) for the Mn(2+)-induced fluorescence change of FITC, though it did shift the IC(50) for the K(+)-induced change. These results suggest that the Mn(2+)-induced fluorescence change is not due to Mn(2+) binding at the transport site. The drawbacks of models in which Mn(2+) stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn(2+) stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.