Autoregulation of the phosphointermediate of Na+/K+-ATPase by the amino-terminal domain of the α-subunit

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na+, K+‐ATPase

“…The stained gels (Fig. 1) confirmed previous observations [6,13], showing that the major product of the cleavage of a-subunit was a 83-kDa peptide whose N-terminal residue has been determined to be Ala 267 [6,14]. The data also showed (Fig.…”

“…3), it was evident that the 83-kDa peptide retains the ability to form the acid-stable phosphointermediate. Although previous studies of our laboratory and others [6,13,15] had shown this property of the 83-kDa fragment, no quantitative data relating the extent of cleavage of a-subunit to the level of phosphoenzyme were reported in these studies. Therefore, in experiments in Fig.…”

“…The Na+-dependent treatment with tx-chymotrypsin [6,9], phosphorylation with [y-32p]ATP in the presence of Na + and Mg 2+ at 0°C [6,7,10], assay of total phosphoenzyme on filters [10], resolution and assays of 32P-labeled peptides on acid gels either by autoradiography [6,7] or by counting of gel slices [11,12], and analysis of N-terminal sequences [6,9] were done by procedures the details of which we have described before. The relative quantities of stained peptide bands on gels were determined by densitometry [7,9].…”

“…4, we exposed Na+,K+-ATPase to chymotrypsin and, at various time intervals, we removed aliquots to assay for the relative amounts of remaining a-subunit, the resulting 83-kDa peptide, and total acid-stable phosphoenzyme formed in the presence of ATP, Mg 2+, and Na +. Phosphorylation conditions were chosen to assure the formation of maximal levels of phosphoenzyme from a-subunit and 83-kDa peptide [6,7]. The results clearly showed that as ~x-subunit decreased and 83-kDa peptide increased with time, there was a time-dependent increase in the level of total phosphoenzyme, followed by a subsequent decline (Fig.…”

“…This controversy which has been reviewed repeatedly [3 5] is caused by disagreements on the extent of contaminants in the commonly used purified preparations of this membrane-bound enzyme and on the relative merits of different methods of protein assay used for the quantitation of enzyme subunits in these preparations. In the course of our recent structure-function studies on Na+,K+-ATPase preparations that are subjected to controlled proteolysis [6][7][8][9], it became evident that determination of relative phosphorylation capacities of the native enzyme and the enzyme whose cc-subunit is cleaved at Leu 266 Ala 267 may resolve the above controversy. Here, we present the results of such determinations, showing that, while this cleavage inhibits enzyme activity, it restores phosphorylation capacity to half of the phosphorylation sites that are dormant in the native enzyme.…”