title: Effect of reducing agents on the solubilization of renal sodium and potassium dependent ATPase with detergent.
abstract: A high concentration (0.5 M) of a reducing agent such as dithiothreitol or 2-mercaptoethanol inactivated both crude (microsomal) and purified preparations of Na+,K+-ATPase only in the presence of detergent. The concentration of SDS needed for inactivation by a reducing agent was low (around 0.3 mg/ml), and was similar to that required for activation in the absence of reduction. The subunits of the enzyme after inactivation with a reducing agent were not sedimented by centrifugation at 100,000 x g for 1 h. Electron microscopic observation of the inactivated enzyme revealed that membranous structures were dominant in the pellet obtained by centrifugation, whereas many particles were present in the supernatant. These results suggest that Na+,K+-ATPase was inactivated by reduction of disulfide bond(s) embedded within the lipid bilayer in the presence of detergent, and that the subunits of the resulting enzyme were no longer bound to the membrane.
mesh_terms: D000818:Animals; D003840:Deoxycholic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004285:Dogs; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology*; D008854:Microscopy, Electron; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D003840:Deoxycholic Acid; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D000254:Sodium-Potassium-Exchanging ATPase; D004229:Dithiothreitol