2 ACTIVE NA+/K+-ATPASES OF HIGH-AFFINITY FOR OUABAIN IN ADULT-RAT BRAIN MEMBRANES

The degree of heterogeneity of active Na+ K+-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+ K+-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 ± 0.15 nM, 460 ± 4.0 nM and 320 ± 4.6 μM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 ± 0.2 nM (very high) and 80 ± 1nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 μM). When the high-affinity sites were involved (ouabain doses lower than 2 μM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 μM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+ K+-ATPase α1, α2 and α3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides. © 1990.