Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase.
Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units-does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K)-ATPase in both soluble and membranous states.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013552:Swine; D013816:Thermodynamics; D013861:Thiocyanates
D005452:Fluoresceins; D046911:Macromolecular Substances; D013861:Thiocyanates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate