METAL-CATALYZED CLEAVAGE OF NA,K-ATPASE AS A TOOL FOR STUDY OF STRUCTURE-FUNCTION RELATIONS

This paper describes a novel technique for specific cleavage of renal Na/K-ATPase, based on bound transition metal ions. The approach might have application to other P-type pumps or membrane proteins. In one ty pe of experiment, specific cleavages of the alpha subunit have been ob served following incubation with ascorbate plus H2O2. Five fragments w ith intact C-terminals and complementary fragments with intact N-termi nals are detectable. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added submicromolar concentrations of Fe2+ ions. The results suggest that Fe2+ (or Fe3+) binds with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. The rate of cleavage is greatly affected by the conformational state of the protein, E1Na or E-2(Rb), respectively. The findings provide information on spatial organizatio n of the protein and suggest that the highly conserved regions of the a subunit, within the minor and major cytoplasmic loops, interact in t he E-2 or E-2(Rb) conformations, but move apart in the E-1 or E1Na con formations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cl eavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proxi mity between trans-membrane segments M5-M10 within the a subunit and f or interacting segments of alpha and beta subunits. We discuss the imp lications of metal-catalysed cleavages for spatial organisation of tra nsmembrane helices of the protein.