A microsomal fraction rich in Na+,K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissuefraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450#umol and 250-290gmol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.

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SUMMARY1. Using a rate-dialysis technique at 0-2 'C, the affinities of Na+ and K+ for the sodium pump of pig kidney outer medulla were determined from their effects on the binding of ADP to the enzyme.2. Since all experiments were carried out in the presence of Tris, the enzyme in absence of its specific ligands was assumed to be in a 'sodium-like' conformation.3. The model used in the analysis of the results assumed the enzyme to be a dimeric structure with two identical high-affinity nucleotide-binding sites. It is concluded from the data that the effects of Na+ and K+ on the binding of nucleotide to either subunit of a nucleotide-free enzyme are identical.4. The two subunits, taken together, have five identical and non-interacting K+-binding sites (KdjSS = 0 5 mM) whose occupation antagonizes nucleotide binding. 7. It is likely that the cation sites investigated are intracellular ones and it is concluded that the binding of each cation to its site induces a specific conformational change in the neighbourhood of the site itselfwithout affecting the regions around the remaining cation binding sites.

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