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 c leavage 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 contamina nt or added Fe2+ ions, as inferred by suppression of cleavages with no nspecific metal complexants (histidine, EDTA, phenanthroline) or the F e3+ 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 ef fects are insensitive to OH . radical scavengers, Cleavages are sensit ive to conformation, In low ionic strength media (E-2) or media contai ning Rb ions [E-2(Rb)], cleavage is much faster than in high ionic str ength media (E-1) or media containing Na ions (E1Na), N-terminal fragm ents and two C-terminal fragments (N-terminals E-214 and V-712) have b een identified by amino acid sequencing. Approximate positions of othe r cleavages were determined with specific antibodies. The results sugg est that Fe2+ (or Fe3+) ions bind with high affinity at the cytoplasmi c surface and catalyze cleavages of peptide bonds close to the Fe2+ (o r Fe3+) ion. Thus, cleavage patterns can provide information on spatia l organization of the polypeptide chain, We propose that highly conser ved regions of the alpha subunit, within the minor and major cytoplasm ic loops, interact in the E-2 or E-2(Rb) conformations but move apart in the E-1 or E1Na conformations, We discuss implications of domain in teractions for the energy transduction mechanism, Fe-catalyzed cleavag es may be applicable to other P-type pumps or membrane proteins.