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 C(12)E(8) 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 (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=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 (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=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.
de Lima Santos
Hérica
H
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-FFCLRP, Universidade de São Paulo-USP, 14040-901-Ribeirão Preto, SP, Brasil.
Fortes Rigos
Carolina
C
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-FFCLRP, Universidade de São Paulo-USP, 14040-901-Ribeirão Preto, SP, Brasil.
Ciancaglini
Pietro
P
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-FFCLRP, Universidade de São Paulo-USP, 14040-901-Ribeirão Preto, SP, Brasil. Electronic address: pietro@ffclrp.usp.br.
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011510:Proteolipids; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D008081:Liposomes; D010714:Phosphatidylethanolamines; D011510:Proteolipids; C020485:proteoliposomes; D015060:1,2-Dipalmitoylphosphatidylcholine; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase