Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.
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.
Lutsenko
S
S
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
Anderko
R
R
Kaplan
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D005973:Glutamine; D001120:Arginine; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D012413:Rubidium