Abstract
In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.
Highlights
From the §Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel and the ‡Membrane Biology Laboratory, Department of Physiology, University of California, Los Angeles and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073
The cleavage pattern of H؉,K؉ATPase by Fe2؉ ions is consistent with the existence of two Fe2؉ sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1
Fe2؉-catalyzed cleavage patterns reveal sequences involved in phosphate, Mg2؉, and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E1 7 E2 transitions, and subconformations induced by H؉,K؉-ATPase-specific ligands such as SCH28080
Summary
In the presence of ascorbate/H2O2, Fe2؉ ions or the ATP-Fe2؉ complex catalyze selective cleavage of the ␣ subunit of gastric H؉,K؉-ATPase. The cleavage pattern of H؉,K؉ATPase by Fe2؉ ions is consistent with the existence of two Fe2؉ sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe2؉ or the ATP-Fe2؉ complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. Fe2؉-catalyzed cleavage patterns reveal sequences involved in phosphate, Mg2؉, and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E1 7 E2 transitions, and subconformations induced by H؉,K؉-ATPase-specific ligands such as SCH28080. E1 and E2 conformations of Hϩ,Kϩ-ATPase have been detected directly by selective tryptic digestion [13], ligand effects on specific inhibitors of Hϩ,Kϩ-ATPase [14, 15], ligand-induced fluorescence changes of fluorescein-labeled protein [16, 17] and eosin noncovalently bound within the nucleotide site [18], or a Kϩ-competitive inhibitor, 1-(2-methylphenyl)-4-methylamino-
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