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 (approximately 9 kDa) 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 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) approximately 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.
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
R
R
Goldshleger
D M
DM
Tal
J
J
Moorman
W D
WD
Stein
S J
SJ
Karlish
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D005971:Glutamates; D018698:Glutamic Acid; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D012413:Rubidium / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013552:Swine; D014357:Trypsin
D005971:Glutamates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D003488:Cyanogen Bromide