Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8.
SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4 degrees C after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4 degrees C. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 microM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 microg/ml) and sodium vanadate (3 microM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphabeta)2 association.
Santos
H L
HL
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
Lamas
R P
RP
Ciancaglini
P
P
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002845:Chromatography; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000201:enzymology*; D010042:Ouabain; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D003902:Detergents; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase