CLONING, EXPRESSION AND CHARACTERIZATION OF 2 PUTATIVE CALCIUM-BINDING SITES IN HUMAN NONERYTHROID ALPHA-SPECTRIN

The C-terminus of alpha-spectrins contains two putative calcium-bindin g sites or EF-hands. To characterize the binding, we have isolated clo nes from a human fetal liver cDNA library and expressed several fragme nts comprising either one or both of these sites. When the isolated cl ones were sequenced, we found that three consecutive nucleotides diffe red compared to the published sequence. The discrepancy affected two c odons in the first of the two putative calcium sites. These codons tra nslated into glutamate and phenylalanine, which are identical to the r esidues present at the same position in other alpha-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 constitut es the only functional calcium-binding site in the C-terminus of human non-erythroid alpha-spectrin. Since peptides comprising the second EF -hand bound calcium nearly as well as intact spectrin, it is also appa rent that the second EF-hand constitutes the major binding site for ca lcium in spectrin. The relative change in negative ellipticity, induce d by the binding of calcium, indicates a dissociation constant of appr oximately 120 mu M.