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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Aug 15;92(17):7936–7940. doi: 10.1073/pnas.92.17.7936

Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.

S Lutsenko 1, R Anderko 1, J H Kaplan 1
PMCID: PMC41261  PMID: 7644516

Abstract

Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.

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Selected References

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