Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic CI subunit of Na+/K+-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1 -12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991 -1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the CI subunit of pork kidney Na+/K+-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K+-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1 -12 as well as the C-terminal peptides 991 -1005 and 1005 -101 6 were found on the cytosolic side. Concanavalin A, however, which interacts with the j3 subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex CO(NH,)~ATP, inactivated Na + /K + -ATPase slowly by interacting with the high-affinity ATP binding .site. The inactivation proceeded at 37°C with an inactivation rate constant of 1.34 x lo-, min-' and with a dissociation constant of 0.62 pM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na +/K +-ATPase by Cr(NH,),ATP, and its H20-substituted derivatives Cr(NH3),(H20)ATP, Cr(NH3)2(H20)zATP and Cr(H20)4ATP was carried out. The substitution of the H 2 0 ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H20),ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(II1) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Naf/K+-ATPase by Cr(II1)-ATP complexes.Inactivation of the enzyme by Rh(HzO),ATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 pM. The observed inactivation rate constant of 2.11 x min-' became higher when Na' or Mg2+ or both were present. The presence of K f however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na' reactivated the Rh(HzO),ATP-inactivated enzyme.Co (