The calcium-activated neutral protease calpain is activated in several pathological conditions. Calpain usually hydrolyses one or only a few peptide bonds in its substrate. One prominent substrate for calpain is spectrin and it has been shown that K K-spectrin is the preferred substrate. We now show that the L L-chain of spectrin is also a substrate for calpain proteolysis, and that the cleavage site in each L L-subunit is located at the very Cterminal part of the molecule. Surprisingly, L LI4 41-spectrin is cleaved at a different site than L LI4 42-and L LII4 41-spectrins despite their high degree of sequence identity.z 1999 Federation of European Biochemical Societies.

The C‐terminus of α‐spectrins contains two putative calcium‐binding sites or EF‐hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other α‐spectrins. In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF‐hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore, it is likely that the second EF‐hand constitutes the only functional calcium‐binding site in the C‐terminus of human non‐erythroid α‐spectrin. Since peptides comprising the second EF‐hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF‐hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 μM.

No abstract

The C-terminus of a-spectrins contains two putative calcium-binding sites or EF-hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other a-spectrins.In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF-hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore, it is likely that the second EF-hand constitutes the only functional calcium-binding site in the C-terminus of human non-erythroid a-spectrin. Since peptides comprising the second EF-hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF-hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 pM.