Purified kidney Na+,K+-ATPase whose α-subunit is cleaved by chymotrypsin at Leu266-Ala267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of α-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α-subunits.
