STUDIES ON CALMODULIN FROM DICTYOSTELIUM DISCOIDEUM

Abstract

Calmodulin is a small Ca('2+)-binding protein that mediates the regulatory effect of Ca('2+) on many biological processes. I have purified and characterized calmodulin from amoebae of the eukaryotic microorganism Dictyostelium discoideum and compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca('2+)-dependent activation of brain cyclic nucleotide phosphodiesterase. Dictyostelium calmodulin also activated erythrocyte (Ca('2+) + Mg('2+))-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmunoassays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins examined.;A rabbit antiserum was raised that is highly specific for Dictyostelium calmodulin. In double immunodiffusion experiments, competition radioimmunoassays showed no cross-reactivity between the antiserum and calmodulins from brain and spinach. The antiserum was used to visualize the distribution of calmodulin in Dictyostelium amoebae by indirect immunofluorescence. Cells were examined under various conditions. In all cases, anti-calmodulin staining was concentrated in the cell cortex. Parallel experiments using a monoclonal antibody against Dictyostelium myosin showed that this protein is also enriched in the cortical region of the cell.;Extracts of Dictyostelium amoebae were examined for the presence of calmodulin-binding proteins by chromatography on a Dictyostelium calmodulin-Sepharose 4B affinity column. A high molecular weight protein that exhibits calcium sensitive binding to calmodulin was identified. The subunit molecular weight of the protein is 265,000. The 265K protein also binds to Dictyostelium F-actin; this interaction does not require calcium. One of the in vivo activities of Dictyostelium calmodulin may be to regulate the 265K-actin interaction and thus affect actin-based motility.