Tris (ultra pure) was from Bio-Lab, Jerusalem

Tris (ultra pure) was from Bio-Lab, Jerusalem. 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 subunit, within the minor and major cytoplasmic WS 12 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. quinol oxidase using Fe-chelate-dependent cleavages (22). We describe here a chance discovery that incubation of renal Na/K-ATPase with ascorbate and H2O2 causes selective cleavages of the subunit. We demonstrate that cleavages depend on the presence of contaminating Fe2+ ions in the media and describe properties of the Fe-catalyzed cleavages. MATERIALS AND METHODS Materials. For SDS/PAGE, all reagents were electrophoresis-grade from Bio-Rad. Tris (ultra pure) was from Bio-Lab, Jerusalem. l(+)-ascorbic acid and 30% H2O2 were from Merck. All other reagents were of analytical grade. Enzyme Preparation. Na/K-ATPase (13C18 units/mg protein) was prepared from pig kidneys, assayed, and stored at ?20C in a solution of 250 mM sucrose, 25 mM histidine (pH 7.4), and 1 mM EDTA [as described by J?rgensen (23)]. Before use, membranes were washed twice and suspended in a solution containing 10 mM Tris?HCl, pH 7.4. Cleavage Reaction. Membrane suspensions (0.1C1 mg/ml), with or without added 30 mM RbCl or NaCl, were incubated at 20C with freshly prepared solutions of 4 mM ascorbate (Tris) plus 4 mM H2O2, without or with added FeSO4 or other metals, in a total volume of 30C40 l. To arrest the reaction, 10 l of 5 mM EDTA or 5-fold concentrated gel sample buffer with 5 mM EDTA was added, and samples were assayed for Na/K-ATPase activity or applied to gels, respectively. Gel Electrophoresis, Blotting to Polyvinylidene Difluoride, Immunoblots, Sequencing. Procedures for running of 10% Tricine ((25), were raised against fragments of trypsinized Na/K-ATPase, 19-kDa membranes (5, 24). Antisera included (= 5)Anti-KETYYBlocked N terminus(D636-R651)-Y1016?8.?33.9? ? 1.5 (various heavy metal chelators were Unc5b added to the medium; 25 mM histidine (Hist) partially WS 12 protected the subunit whereas 2 mM EDTA or 1 mM phenanthroline (Phen) completely prevented cleavage. The result indicated the requirement for a heavy metal, which is either tightly bound to the enzyme or is present as a contaminant in the medium. The following experiment showed that the first possibility is unlikely. We incubated the enzyme in a medium containing 4 mM ascorbate/H2O2 and WS 12 30 mM EDTA(Tris) so as to release and chelate any bound ions, washed the enzyme twice in 10 mM Tris?HCl to remove EDTA, and then reincubated with ascorbate/H2O2. The enzyme was cleaved to the same extent as control enzyme (not shown). Analysis of heavy metal content of other components showed that a medium consisting of 4 mM ascorbic acid, 4 mM H2O2 and 10 mM Tris?HCl contains: Fe, 47.5 nM; Ni, 14.4 nM; Cu, 8 nM; Mn, 3.3 nM; Cr, 1.12 nM; Mo, 1.1 nM; Zn, 0.9 nM; and Sn, 0.8 nM. One approach to identify the relevant contaminant metal ion has been to use metal-specific complexants. The Fe3+-selective complexant desferrioxamine suppresses cleavage at 10C100 M (Fig. ?(Fig.22shows that addition of 0.25C10 M Fe2+ ions greatly amplifies the extent of cleavage. Note that the same five cleavages appear as in the absence of added Fe2+ ions and that the cleavages.