Purification and characterization of (Na+ + K+)-ATPase. VI. Differential tryptic modification of catalytic functions of the purified enzyme in presence of NaCl and KCl.

1. Two distinct patterns of tryptic modification of the catalytic functions of purified (Na+ + K+)-ATPase can be related to the two previously described patterns of enzyme inactivation and cleavage of the large chain seen with NaCl and KCl (Jorgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415). 2. With NaCl, in phase A, the rapid inactivation of 50-55% of the (Na+ + K+)-ATPase activity is associated with loss of 85% of the K+-phosphatase activity and an increase in Na+-ADP-ATP exchange activity to 150% of control. ATP binding and phosphorylation are unchanged and the inactivation may result from cleavage of bonds within the large chain which are involved in dephosphorylation reactions. In phase B with NaCl, ATP binding and phosphorylation are lost slowly in parallel to inactivation of (Na+ + K+)-ATPase and cleavage of the large chain to a fragment with Mr=78 000. 3. With KCl, cleavage of the large chain to almost equal fragments abolish ATP binding and phosphorylation in parallel to the inactivation of (Na+ + K+)-ATPase. An additional split seems required for inactivation of the K+-pNPPase activity. 4. After completion of the digestion in phase A with NaCl a stable preparation can be isolated in which the activity of (Na+ + K+)-ATPase is 40%. ATP binding and phosphorylation are 90%, K+-phosphatase is 15%, and Na+-ADP-ATP exchange is 150% of control. We currently examine if these levels are related to changes in phosphorylation kinetics. 5. The ATP binding area is much more stable to trypsin with NaCl than with KCl, but loss of the binding capacity is in both cases correlated to a distinct cleavage of the large chain. The relationship between the fractional loss of ATP binding and cleavage of the large chain suggests that the nucleotide binding area is confined to one of the two large chains in the protein complex with Mr=270 000 which binds one molecule of ATP. 6. The data also suggest that the phosphatase site is remote from the ATP binding area. It is proposed that the protein complex with Mr=270 000 contains two large chains with different catalytic functions and that each chain forms a cation channel.