Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain.
Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology*; D010042:Ouabain / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D011510:Proteolipids / Q000302:isolation & purification*; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*; D014316:Tritium
C017917:2-nitro-5-azidobenzoylouabain; D000596:Amino Acids; D011510:Proteolipids; D014316:Tritium; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase