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

Abstract

Purified kidney Na+,K+-ATPase whose a-subunit is cleaved by chymotrypsin at Leu~66-Ala 267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of s-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 ~-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of c¢-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 c¢-subunits.Key words: Na+,K+-ATPase; Phosphoenzyme; Half-site reactivity; Oligomeric structure; Chymotrypsin Materials and methodsPurified membrane-bound Na+,K+-ATPase of canine kidney medulla, with specific activity in the range of 1000-1600 ktmol of ATP hydrolyzed/mg/h, was prepared and assayed as indicated [9,10]. 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]. The total amount of enzyme protein applied to each gel was in the range of 1 10 I.tg. It was established experimentally that under these conditions there were linear relationships between the amount of native or cleaved enzyme applied to the gel and the densities of stained a-subunit, ~-subunit, and 83-kDa peptide bands. Results

Half-site Modification of Lys-480 of the Na+,K+-ATPase α-Chain with Pyridoxal 5′-Diphospho-5′-adenosine Reduces ATP-dependent Phosphorylation Stoichiometry from Half to a Quarter

“…Chymotryptic digestion increased in the amount of phosphoenzymes (41). These data can be explained, based on full-site phosphorylation, which occurred under those conditions as the result of a decrease or disruption of subunit-interactions (23,31,41).…”

Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K‐ATPase α‐subunit: identification of the N‐terminal fragments containing the transmembrane H1‐H2 domain

“…For example, while the phosphorylation of the native enzyme by ATP is dependent on Na + , the chymotrypsin‐digested enzyme can be phosphorylated in the absence of Na + [6]; and while the phosphorylation by ATP and ADP‐ATP exchange activity of the native enzyme are inhibited by ouabain, these activities of the chymotrypsin enzyme are stimulated by ouabain [6]. Also, our recent studies have established that the dormant half of the phosphorylation sites of the native enzyme are unmasked upon cleavage of the α‐subunit by chymotrypsin [10]. Clearly, the determination of the structural basis of these contrasting properties of the native and the chymotrypsin‐digested preparations is important to the clarification of the relation of the enzyme's structure to its reaction mechanism.…”

Evidence for the Existence of Two ATP-sensitive Rb+Occlusion Pockets within the Transmembrane Domains of Na+/K+-ATPase

“…This is not a likely possibility. Although the existence of two functionally dissimilar ␣-subunits in the native enzyme seems to be established by our recent studies (23), this asymmetry is most likely due to ␣-␣ interactions (19,20,24). However, structural regions of the ␣-subunit on the large intracellular central loop that are presently known to be involved in ␣-␣ contact (24 -26) are no longer present in the digested enzyme, and electron microscopic studies on the digested enzyme also suggest the absence of ␣-␣ associations in this preparation (27).…”