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Location of Asn831 of the alpha chain of Na/K-ATPase at the cytoplasmic surface. Implication for topological models.

Authors
  • Karlish, S J
  • Goldshleger, R
  • Jørgensen, P L
Type
Published Article
Journal
The Journal of biological chemistry
Publication Date
Feb 15, 1993
Volume
268
Issue
5
Pages
3471–3478
Identifiers
PMID: 8381430
Source
Medline
License
Unknown

Abstract

The principal objective of this work has been to determine whether Asn831 of the alpha chain of Na/K-ATPase is located at the cytoplasmic or extracellular surface. Asn831 is the N-terminal residue of a 19-kDa C-terminal tryptic fragment which, together with smaller (8-11 kDa) membrane-embedded fragments of the alpha chain and a largely intact beta chain, is produced by extensive tryptic digestion of renal Na,K-ATPase in the presence of Rb+ and absence of Ca2+ ions. In these so-called "19-kDa membranes," Rb+ and Na+ occlusion capacities are normal, but ATP-dependent activities are lost. Transmembrane segments in the 19-kDa and other fragments are thought to contribute ligating residues to a cation occlusion "cage." However, the arrangement of transmembrane segments of the alpha chain is uncertain, particularly in the critical C-terminal domain. Different topological models predict that the N-terminal Asn831 of the 19-kDa fragment lies at opposite surfaces, respectively. The location of Asn831 has been studied by tryptic digestion of either tight right side-out-oriented membrane vesicles isolated from renal medulla or phospholipid vesicles reconstituted with renal Na/K-ATPase, containing pumps in both right side-out and inside-out orientations. Digestion is performed in the presence of Rb+ and absence of Ca2+ ions. In native renal membrane vesicles the alpha chain is not split by trypsin. By contrast, the beta chain is partially split to a fragment of 16 kDa, beginning at the N-terminal Ala. This fragment is 4 residues longer than a 16-kDa fragment produced by digestion of purified Na/K-ATPase, a finding which shows that trypsin has full access to the extracellular surface, but not to the interior of the vesicles. In the presence of deoxycholate, which destroys the membrane permeability barrier, trypsin digests the alpha chain and the 19-kDa fragment (N-terminal Asn831) is produced. Extensive tryptic digestion of phospholipid vesicles reconstituted with the Na/K-ATPase leads to digestion of the alpha chain and appearance of intermediate fragments and then the 19-kDa fragment. About 25% of the alpha chains in the preparation are resistant to digestion. Functional studies show that the trypsin-resistant pumps are in the right side-out orientation. Observed tryptic fragments were sequenced and the following N termini were detected: Glu31, 33 kDa; Ser221, 78 kDa; Ile470, 56 kDa; Ala590, 39 kDa; and Asn831, 19 kDa. Observation of these N termini is consistent with digestion of the alpha chain of inside-out-oriented pumps at cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)

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