Chemical modification of Glu-953 of the α chain of Na⁺,K⁺-ATPase associated with inactivation of cation occlusion

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na⁺,K⁺-ATPase, using the carboxyl reagent N,N′-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na⁺,K⁺-ATPase - the so-called "19-kDa membranes" - containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the α chain, and a largely intact β chain; these membranes have normal Rb⁺ and Na⁺ occlusion capacities. The 19-kDa peptide and a smaller (≈9 k Da) unidentified peptide(s) are labeled by [¹⁴C]DCCD in a Rb⁺-protectable fashion. Rb⁺-protected [¹⁴C]DCCD incorporation into the "19 kDa membranes" and into native Na⁺,K⁺-ATPase is linearly correlated with inactivation of Rb⁺ occlusion. Similar linear correlations are observed when Rb⁺-protected [¹⁴C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of a chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb⁺ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per a chain. We suggest that each of two K⁺ (or Na⁺) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the a chain. After cyanogen bromide digestion of purified, labeled a chain, or of 19-kDa peptide, a labeled fragment of apparent M_r ≈4 kDa was detected and was identified as that with NH₂-terminal Lys-943. Rb⁺-protected [¹⁴C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K⁺ (or Na⁺) sites, but only neutral groups in the third Na⁺ site.