Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.
In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity.
Instituto de Física da Universidade de São Paulo, IF USP, 05508-090 São Paulo, Brazil; Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, FFCLRP USP, 14040-901 Ribeirão Preto, SP, Brazil.
Juliana Sakamoto
JS
Yoneda
Instituto de Física da Universidade de São Paulo, IF USP, 05508-090 São Paulo, Brazil.
Gustavo
G
Scanavachi
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, FFCLRP USP, 14040-901 Ribeirão Preto, SP, Brazil.
Heitor Gobbi
HG
Sebinelli
Instituto de Química de São Carlos, IQSC-USP, 13560-970 São Carlos, SP, Brazil.
Júlio Cesar
JC
Borges
Instituto de Física da Universidade de São Paulo, IF USP, 05508-090 São Paulo, Brazil.
Leandro R S
LR
Barbosa
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, FFCLRP USP, 14040-901 Ribeirão Preto, SP, Brazil.
Pietro
P
Ciancaglini
Instituto de Física da Universidade de São Paulo, IF USP, 05508-090 São Paulo, Brazil. Electronic address: itri@if.usp.br.
Rosangela
R
Itri
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D007679:Kidney Medulla / Q000737:chemistry; D007700:Kinetics; D008027:Light; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization; D011817:Rabbits; D053838:Scattering, Small Angle; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility
D003902:Detergents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000254:Sodium-Potassium-Exchanging ATPase
Equilibrium state; Na-K,ATPase; Oligomerization