CHEMICAL MODIFICATION OF GLU-953 OF THE ALPHA-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 alpha-chain, and a largely intact beta-chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (almost-equal-to 9 kDa) unidentified peptide(s) are labeled by [C-14]DCCD in a Rb+-protectable fashion. Rb+-protected [C-14]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 [C-14]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha-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 alpha-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 alpha-chain. After cyanogen bromide digestion of purified, labeled alpha-chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) almost-equal-to 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb+-protected [C-14]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.