Kinetics behaviors of Na,K-ATPase: Comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C12E8 solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na+-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K0.5 = 6 μM and 4 μM for reconstituted enzyme and purified form, respectively) and another of low affinity (K0.5 = 0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (αβ)2 oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg2+ concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K0.5 value of 0.4 mM. The Na+ ions also presented a single saturation curve with VM = 551 U/mg and K0.5=0.2 mM with cooperative effects. The activity was also stimulated by K+ ions through a single curve of saturation sites (K0.5 = 2.8 mM), with cooperative effects and VM = 641 U/mg. The lipid microenvironment close to the proteic structure and the K+ internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.