Stabilization and enhancement of primary cytostatic factor (CSF) by ATP and NaF in amphibian egg cytosols.

Amphibian zygotes microinjected with the cytoplasm or cytosol of unactivated eggs are arrested at metaphase of mitosis. The activity responsible for this effect has been designated primary "cytostatic factor (CSF)." Primary CSF disappears from the cytoplasm after egg activation, as well as from cytosols after addition of Ca2+. In the present study, using fresh cytosols of Rana pipiens eggs, a unit of CSF activity was defined as the dose required to arrest 50% of the recipients, and the specific activity of a cytosol was expressed in units per microgram protein. Specific activities of cytosols prepared with the one-step centrifugation method employed in the present study were double the activities in cytosols obtained by the previously described two-step procedure. During storage at 2 degrees C, CSF specific activity in cytosols fell rapidly within hours of extraction and disappeared completely within 2 days. However, if NaF and ATP were added to fresh cytosols, specific activities increased within hours and remained high for at least several days. Addition of gamma-S-ATP also significantly increased the longevity of the activity during storage at 2 degrees C. Further, it was found that primary CSF activity could be recovered by ATP additions to cytosols in which residual activity was still present, but no activity was recovered by ATP addition if cytosols had completely lost activity. When Ca2+ was added to cytosols to which NaF and ATP had been added, CSF was inactivated more slowly than in control cytosols without NaF and ATP additions. Therefore, it appears that maintenance of primary CSF activity in vitro requires protein phosphorylation and that protein dephosphorylation is involved with its inactivation. Also, we compared the sensitivities to primary CSF of Xenopus laevis and R. pipiens two-cell embryos. In order to arrest 50% of recipients, the concentration of primary CSF in Xenopus blastomeres was three times higher than in Rana blastomeres.