Calcium binding to membrane skeletal proteins

Abstract

Some cytoskeletal proteins have been found to bind calcium (Anthony and Babitch, 1984). We investigated the calcium binding properties of brain tubulin, erythrocyte and brain spectrins, and muscle and platelet tropomyosins. Brain tubulin was found to have 2 classes of calcium binding sites. There are 1.56 high affinity sites with 4.86$\mu$M dissociation constant and 5.82 low affinity sites of 64$\mu$M dissociation constant. In the presence of 60$\mu$M magnesium, the high affinity sites reduce to 0.64 sites with 4.7$\mu$M dissociation constant and only 1.2 low affinity sites with 36uM dissociation constant remain. This suggests that there are both 'Ca-specific' and 'Ca-Mg' (or divalent cations) sites present which may perform regulatory and structural roles respectively (Robertson et al., 1981). Enzyme cleavage data show the calcium binding sites to be at the C-terminal of each subunit. Both erythrocyte and brain spectrins bind calcium specifically with high affinity. There are 5 binding sites per erythrocyte dimer with dissociation constant of 5$\mu$M. Brain spectrin tetramers contain 10 calcium binding sites of about 5$\mu$M dissociation constant. The presence of 0.5mM magnesium does not abolish calcium binding which shows that the binding is calcium specific. The binding is not to phosphate groups because both subunits bind calcium equally on a protein basis and spectrin degradation products which lose the phosphorylation sites were observed to bind calcium. 5-15% linear sucrose gradient and native gel data suggest the binding of calcium probably disturbs the dimer-tetramer equilibrium favouring dimer formation. Circular dichroism data shows no significant changes of spectrin secondary structure upon binding calcium. Muscle and platelet tropomyosins both bind calcium with high affinity. There are about 2 binding sites with less than 1$\mu$M dissociation constant. These sites are divalent cation sites because the presence of 0.5mM magnesium abolishes the binding. Enzymatic and chemical cleavage data suggest that the binding sites are probably in the middle, non-rigid region around cys-190.