Metal-catalysed 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 type of experiment, specific cleavages of the alpha subunit have been observed following incubation with ascorbate plus H2O2. Five fragments with intact C-terminals and complementary fragments with intact N-terminals 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 E2(Rb), respectively. The findings provide information on spatial organization of the protein and suggest that the highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations, but move apart in the E1 or E1Na conformations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cleavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proximity between trans-membrane segments M5-M10 within the alpha subunit and for interacting segments of alpha and beta subunits. We discuss the implications of metal-catalysed cleavages for spatial organisation of transmembrane helices of the protein.
Goldshleger
R
R
Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Bar Shimon
M
M
Or
E
E
Karlish
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D003300:Copper / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D008670:Metals / Q000494:pharmacology*; D008968:Molecular Conformation; D017433:Protein Structure, Secondary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine
D005290:Ferric Compounds; D005296:Ferrous Compounds; D008670:Metals; D003300:Copper; D000254:Sodium-Potassium-Exchanging ATPase