Unraveling the Na,K-ATPase alpha(4) subunit assembling induced by large amounts of C(12)E(8) by means of small-angle X-ray scattering.
In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alphabeta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.
Barbosa
Leandro Ramos Souza
LR
Instituto de Física da Universidade de São Paulo, Cx. Postal 66318, CEP 05315-970 - São Paulo, SP, Brazil. lbarbosa@if.usp.br
Rigos
Carolina Fortes
CF
Yoneda
Juliana Sakamoto
JS
Itri
Rosangela
R
Ciancaglini
Pietro
P
D000818:Animals; D003902:Detergents / Q000737:chemistry*; D007679:Kidney Medulla / Q000201:enzymology; D008823:Micelles; D011092:Polyethylene Glycols / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry; D011817:Rabbits; D053838:Scattering, Small Angle; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
D003902:Detergents; D008823:Micelles; D021122:Protein Subunits; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000254:Sodium-Potassium-Exchanging ATPase