Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide.

In addition to the (Na(+)+K(+))ATPase another P-ATPase, the ouabain-insensitive Na(+)-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg(2+), MgATP and furosemide on the Na(+)-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na(+)-ATPase at pH 7.0 are: (a) K(0.5) for Na(+)=8.9+/-2.2mM; (b) K(0.5) for MgATP=1.8+/-0.4mM; (c) two sites for free Mg(2+): one stimulatory (K(0.5)=0.20+/-0.06 mM) and other inhibitory (I(0.5)=1.1+/-0.4 mM); and (d) I(0.5) for furosemide=1.1+/-0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na(+) (K(0.5)=19.5+/-0.4) and MgATP (K(0.5)=3.4+/-0.3 mM) but increases the apparent affinity for furosemide (0.18+/-0.02 mM) and Mg(2+) (0.05+/-0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na(+) (K(0.5)=18.7+/-1.5 mM) and furosemide (I(0.5)=3.04+/-0.57 mM) but does not change the apparent affinity to MgATP and Mg(2+). The data presented in this paper indicate that the modulation of the Na(+)-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg(2+) and MgATP complex may play an important role in the Na(+)-ATPase physiological regulatory mechanisms.