Fe-catalyzed cleavage of the alpha subunit of Na/K-ATPase: evidence for conformation-sensitive interactions between cytoplasmic domains.
Incubation of Na/K-ATPase with ascorbate plus H2O2 produces specific cleavage of the alpha subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe2+ ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe3+-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe2+ but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH. radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E2) or media containing Rb ions [E2(Rb)], cleavage is much faster than in high ionic strength media (E1) or media containing Na ions (E1Na). N-terminal fragments and two C-terminal fragments (N-terminals E214 and V712) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe2+ (or Fe3+) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations but move apart in the E1 or E1Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.
Goldshleger
R
R
Biochemistry Department, Weizmann Institute of Science, Rehovot, 76100, Israel.
Karlish
S J
SJ
D000818:Animals; D007501:Iron; D008958:Models, Molecular; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine
D007501:Iron; D000254:Sodium-Potassium-Exchanging ATPase