Specific Cu2+ -catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface

This paper describes specific Cu 2+ -catalyzed oxidative cleavage of α and β subunits of Na,K-ATPase at the extracellular surface. Incubation of right side-out renal microsomal vesicles with Cu 2+ ions, ascorbate, and H 2 O 2 produces two major cleavages of the α subunit within the extracellular loop between trans-membrane segments M7 and M8 and L7/8. Minor cleavages are also detected in loops L9/10 and L5/6. In the β subunit two cleavages are detected, one before the first S-S bridge and the other between the second and third S-S bridges. Na,K-ATPase and Rb + occlusion are inactivated after incubation with Cu 2+ /ascorbate/H 2 O 2. These observations are suggestive of a site-specific mechanism involving cleavage of peptide bonds close to a bound Cu 2+ ion. This mechanism allows several inferences on subunit interactions and spatial organization. The two cleavage sites in L7/8 of the α subunit and two cleavage sites of the β subunit identify interacting segments of the subunits. L7/8 is also close to L9/10 and to cation occlusion sites. Comparison of the locations of Cu 2+ -catalyzed cleavages with Fe 2+ -catalyzed cleavages (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596–9601) suggests division of the membrane sector into two domains comprising M1–M6 and M7–M10/Mβ, respectively.