The association of Na,K-ATPase subunits studied by circular dichroism, surface tension and dilatational elasticity.
Different stoichiometries are observed between alpha and beta subunits of Na,K-ATPase that depend on the method employed to solubilize and purify the enzyme. It is not known whether this variability is due to loss of protein-protein association, or is a result of the replacement of essential phospholipids by detergent molecules. With the aim of understanding the effect of enzyme/surfactant ratio on both the catalytic activity and the enzyme structure, we have investigated the bulk and surface properties of the enzyme. The circular dichroism (CD) spectra, surface tension and dilatational surface elasticity results were compared with the residual ATPase activity of the Na,K-ATPase in different surfactant and protein concentrations. Na,K-ATPase in the (alphabeta)(2) form dissociated to the alphabeta form on dilution, and associated to the (alphabeta)(4) form when concentrated. These different stoichiometries have similar ATPase activities and are in equilibrium at C(12)E(8) concentrations below the CMC (0.053 mg mL(-1)). At detergent concentrations above the CMC the ATPase activity of all forms was abolished, which is concomitant with the dissociation of the alpha and beta subunits.
Rigos
Carolina Fortes
CF
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto SP, Brazil.
Nobre
Thatyane Morimoto
TM
Zaniquelli
Maria Elisabete Darbello
ME
Ward
Richard John
RJ
Ciancaglini
Pietro
P
D000818:Animals; D002942:Circular Dichroism; D004548:Elasticity; D007679:Kidney Medulla / Q000201:enzymology; D008823:Micelles; D011092:Polyethylene Glycols; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry*; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013500:Surface Tension; D013501:Surface-Active Agents
D008823:Micelles; D021122:Protein Subunits; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D000254:Sodium-Potassium-Exchanging ATPase