title: Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro.
abstract: Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass = 21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16 +/- 1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of mu- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.
authors.affiliation: Department of Biochemistry, University of Oxford, UK. eschrode@ex.ac.uk
authors.forename: E
authors.initials: E
authors.lastname: Schröder
authors.forename: A C
authors.initials: AC
authors.lastname: Willis
authors.forename: C P
authors.initials: CP
authors.lastname: Ponting
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D019281:Dimerization; D004912:Erythrocytes / Q000378:metabolism; D006360:Heat-Shock Proteins; D008969:Molecular Sequence Data; D010544:Peroxidases; D054464:Peroxiredoxins; D016415:Sequence Alignment; D013379:Substrate Specificity; D013552:Swine
chemical_list: D001798:Blood Proteins; D006360:Heat-Shock Proteins; D010544:Peroxidases; C086065:PRDX2 protein, human; D054464:Peroxiredoxins; D002154:Calpain