CYTOPLASMIC DYNEIN OF THE SEA URCHIN EGG I. PARTIAL PURIFICATION AND CHARACTERIZATION

Cytoplasmic ATPase of sea urchin eggs was partially purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel-filtration chromatography and sucrose density gradient centrifugation. The specific activity increased to 0.7 μmole/min/mg protein indicating 100 fold purification. The ATPase had a sedimentation constant of 12S and was highly specific for ATP. The enzyme fraction contained neither (Na, K)-ATPase, Ca-ATPase, oligomycin-sensitive ATPase, phosphatases, nor myosin. This cytoplasmic ATPase was inhibited by a low concentration of vanadate (V). Half-maximal inhibition was observed at a vanadate concentration of 1 μM at low ionic strength. The inhibition was almost totally reversed by addition of norepinephrine. The vanadate-sensitivity of cytoplasmic ATPase decreased with increasing KCl concentration. The activation by Mg2+ or Ca2+ , and dependence of the activity on KCl concentration characteristic of dyneins from sea urchin sperm flagella and the embryonic cilia were observed with cytoplasmic ATPase. These results allowed the cytoplasmic ATPase to be classified as a dynein. In addition, this designation was reinforced by the fact that an oligomeric 23S form of cytoplasmic dynein was identified in the cytoplasm as well as in the isolated mitotic apparatus.