Glutamate 779, an intramembrane carboxyl, is essential for monovalent cation binding by the Na,K-ATPase.

Abstract

Incubation of purified renal Na,K-ATPase with the fluorescent carboxyl-selective reagent, 4-(diazomethyl)-7-(diethylamino)- coumarin (DEAC), results in enzyme inactivation via disruption of the monovalent cation binding sites and loss of K+ and Na+ binding capacity. Modification of 1 or 2 carboxyl residues in the alpha-subunit in a K+ or Na(+)-preventable manner leaves the ATP binding unaltered, and the enzyme is still able to undergo the major conformational transitions (Argüello, J. M., and Kaplan, J. H. (1991) J. Biol. Chem. 266, 14627-14635). Labeled alpha-subunits of Na,K-ATPase were isolated by gel electrophoresis and digested using V8 protease. The digests contained two bands which were fluorescently labeled in a cation-protectable fashion. Four peptides were identified in these bands. Peptides I (apparent molecular mass approximately 17 kDa), II (approximately 15 kDa), and IIIa (approximately 5 kDa) start at Gly758 while Peptide IIIb (approximately 5 kDa) starts at Gly561. Subsequent proteolysis of peptides IIIa and IIIb with thermolysin followed by electrophoresis revealed a single smaller fluorescent peptide which passed through 3-kDa cut-off membrane filters but was retained by 1-kDa cut off filters. N-terminal sequence analysis of this peptide gave the sequence 773Leu-Thr-Ser-Asn-Ile-Pro-Glu-Ile-Thr-Pro-Phe-Leu. The length of this peptide was also examined in labeling experiments with cysteine-reactive probes which indicated that the peptide did not extend to the next carboxyl-containing amino acid residue in the alpha-subunit sequence (Asp804). The site of attachment of DEAC is thus Glu779, an intramembrane carboxyl residue present in all known sequences of alpha-subunit isoforms of the Na,K-ATPase. This glutamate is essential for Na+ and K+ binding and active transport by the sodium pump. Its location in the fifth transmembrane segment provides a way in which information about ATP binding and phosphorylation in the major cytoplasmic loop of the enzyme is transmitted to intramembrane cation sites during the reaction cycle.