A Ca-activated ATPase in the mitotic apparatus of the sea urchin egg (isolated by a new method)

Abstract

Steps in a new procedure for isolating the mitotic apparatus from sea urchin eggs ( Strongylocentrotus purpuratus) are: (1) Cultivation of the eggs in sea water in which Na is replaced by Li, under which conditions the MA is stabilized in vivo and does not break down at the end of mitosis; (2) storage of the eggs containing the MA in 30% ethanol at −10 °C, preserving isolability and ATPase activity for several months; (3) isolation of the MA in the presence of ethanol and Triton X-100 at +10 °C by selective dispersal of the cytoplasm; (4) purification by washing in 30% ethanol, 0.1% Triton at low temperature. Because of the stabilization of the MA in the Li-sea water, the yield is large. A Ca-activated ATPase, described in detail by Petzelt [22], is concentrated in the MA at specific activities 2–3 times those measured in the cytoplasm. Acrylamide gel electrophoresis shows that the subunits of microtubule protein are major components of the MA prepared by the present method.