Sidedness of the effect of amines on the steady-state phosphorylation level of reconstituted Na+/K+-ATPase.

The sidedness of the effects of several amines on the steady-state phosphorylation level of rabbit kidney Na+/K+-ATPase has been studied with the enzyme incorporated in phosphatidylcholine-cholesterol containing proteoliposomes. The presence of ouabain prevented phosphorylation of non-incorporated or rightside-out incorporated enzyme, so that only the inside-out incorporated Na+/K+-ATPase molecules were studied. Addition of either Na+ or several amines to the extracellular side of the enzyme led to an enhancement of the steady-state phosphorylation level obtained with optimal concentrations of Na+, Mg2+ and ATP at the cytosolic side. The series imidazole greater than Na+ greater than triallylamine greater than Tris greater than ethylenediamine showed a decrease in affinity. Histidine, sorbitol and choline chloride had no effect at the extracellular side. This means that in addition to the well-known cytosolic ligands either Na+ or a positively charged amine buffer has to be present extracellularly in order to obtain an optimal phosphorylation level. At the cytoplasmic side the tested amines exerted different effects. (i) Imidazole and triallylamine enhanced the steady-state phosphorylation level when the extracellular conditions were optimal (saturating amine concentration). (ii) Tris and ethylenediamine decreased the steady-state phosphorylation level and (iii) histidine had no effect. The cytoplasmic effects of the amine compounds correlate with those described by Schuurmans Stekhoven et al. (Biochim. Biophys. Acta 937 (1988) 161-171) for the unsided preparation. The extracellular effects, however, are apparently masked in experiments with fragmented enzyme preparations and are assumed to be potentiating effects which make the enzyme ready for phosphorylation upon a cytoplasmic trigger (e.g. Na+).