Two 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 microM, 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 +/- 1 nM (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 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) 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 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.
Berrebi-Bertrand
I
I
INSERM U127, Hôpital Lariboisière, Université Paris 7, France.
Maixent
J M
JM
Christe
G
G
Lelièvre
L G
LG
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000201:enzymology* / Q000254:growth & development; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase