Specific Cu2+-catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface.
This paper describes specific Cu2+-catalyzed oxidative cleavage of alpha and beta subunits of Na,K-ATPase at the extracellular surface. Incubation of right side-out renal microsomal vesicles with Cu2+ ions, ascorbate, and H2O2 produces two major cleavages of the alpha 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 beta 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 Cu2+/ascorbate/H2O2. These observations are suggestive of a site-specific mechanism involving cleavage of peptide bonds close to a bound Cu2+ ion. This mechanism allows several inferences on subunit interactions and spatial organization. The two cleavage sites in L7/8 of the alpha subunit and two cleavage sites of the beta 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 Cu2+-catalyzed cleavages with Fe2+-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/Mbeta, respectively.
Shimon
M B
MB
Biochemistry Department, Weizmann Institute of Science, Rehovot 76100, Israel.
Goldshleger
R
R
Karlish
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003300:Copper / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology*; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D046911:Macromolecular Substances; D010446:Peptide Fragments; D003300:Copper; D007501:Iron; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium