MEMBRANE DISPOSITION OF THE M5-M6 HAIRPIN OF NA+,K+-ATPASE ALPHA SUBUNIT IS LIGAND DEPENDENT

摘要

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 submit 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. [References: 26]