The phosphorylated intermediate in the (Na + K)-activated adenosine triphosphatase (Na-K ATPase) has been characterized as an L-glutamyl-γ-phosphate residue in the enzyme. This has been accomplished by digestion of the phosphorylated and nonphosphorylated forms of the enzyme with pepsin, reaction of the pepsin digests with [2,3-3H]N-(n-propyl)hydroxylmine, further digestion of the derivatized peptides with pronase in the presence of carrier L-glutamyl-γ-N-(n-propyl)hydroxamate and carrier L-aspartyl-N-(n-propyl)hydroxamate, and chromatographic purification. An increment in radioactivity migrated with authentic L-glutamyl-γ-N-(n-propyl)hydroxamate in a total of seven electrophoretic and chromatographic systems and on gel filtration. No increment in radioactivity was associated with authentic L-aspartyl-ß-N-(n-propyl)hydroxamate in five out of the seven chromatographic and electrophoretic systems. At the last stage of purification the radioactivity from the phosphorylated enzyme which migrated as L-glutamyl-γ-N-(n-propyl)hydroxamate was 2½ times that from the nonphosphorylated enzyme. On the basis of these results it is concluded that the phosphorylated intermediate in the Na-K ATPase is an L-glutamyl-γ-phosphate residue. The beef brain Na-K ATPase has been solubilized with the nonionic detergent, Lubrol, and has been purified 10 times over that in the original microsomes. The soluble enzyme remains stable in the presence of ATP and either Na+ or K+. If the partially purified enzyme is electrophoresed in 3% polyacrylamide, followed by incubation with ATP, Na+, K+, and Mg++, a single, somewhat diffuse, ATPase band, which is ouabain-sensitive is seen. Protein impurities are also seen on the gel. Gel electrophoresis, after treatment of the partially purified enzyme with phenol-acetic acid-urea, shows about 12 discrete protein bands. Studies on the site-directed alkylation of the (Na + K)-activated adenosine triphosphatase with haloacetate derivatives of cardiotonic steroids are reviewed. Efforts are now underway to specifically alkylate the cardiotonic steroid site of the Na-K ATPase with hellebrigenin 3-[2-3H]iodoacetate and to purify the subunit of the enzyme containing the cardiotonic steroid site by following radioactivity. Finally, a working model for the role of the Na-K ATPase in the coupled transport of Na and K is presented.

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