ABSTRACT
This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure-function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP-Fe2+, which directs the Fe2+ to the normal ATP-Mg2+ site. Fe2+- and ATP-Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane-water interface. Cu2+ or Cu2+-DPP-catalyzed cleavages are providing information on alpha/beta subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.
Subject(s)
Energy Transfer/physiology , Metals/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Catalysis , Cell Membrane/metabolism , Cytoplasm/metabolism , Magnesium/chemistry , Models, Molecular , Molecular Conformation , Oxidation-Reduction , Phosphorus/chemistry , Structure-Activity RelationshipABSTRACT
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.
