Two Ca2(+)-binding proteins in the mitotic apparatus of sea urchin eggs

Two Ca2(+)-binding proteins of sea urchin eggs were purified and partially characterized. They showed Ca2(+)-dependent binding to actin filaments and Ca2(+)-dependent changes of fluorescence intensity which was used to estimate the affinity constant of these proteins to Ca2+ ions. Ca2+ ions did not increase phospholipid binding ability of these proteins. Therefore these proteins are distinguished from the calpactin family. Staining of sections of metaphase eggs embedded in paraffin showed their localization in the mitotic apparatus. Furthermore, staining of whole mount eggs with anti-tubulin and antibodies against these proteins, followed by observations with confocal laser-scanning microscopy showed their co-localization with microtubules more clearly. In vitro co-sedimentation assay of microtubules with these proteins, however, showed no interaction between them. This suggested that some structures surrounding the mitotic apparatus microtubules are responsible for their localization.