Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro

E Schröder, A C Willis, C P Ponting

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)279-91
Number of pages13
JournalBBA - Bioenergetics
Volume1383
Issue number2
Publication statusPublished - 2 Apr 1998

Keywords

  • Amino Acid Sequence
  • Animals
  • Blood Proteins
  • Calpain
  • Dimerization
  • Erythrocytes
  • Heat-Shock Proteins
  • Molecular Sequence Data
  • Peroxidases
  • Peroxiredoxins
  • Sequence Alignment
  • Substrate Specificity
  • Swine

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