Fractionation of cytostatic factors from cytosols of amphibian eggs.
The cytoplasm or cytosols of unfertilized amphibian eggs contain cytostatic factor (CSF) which arrests cleavage at metaphase after injection into a zygote. After addition of Ca2+ to cytosols, the initial CSF (CSF-1) is inactivated, yet CSF develops again during storage at 2 degrees C (CSF-2). We have separated the two CSFs by ultracentrifugation and ammonium sulfate fractionation using Rana pipiens egg cytosols. CSF-1 was sedimented by ultracentrifugation. After Ca2+ addition, the lighter fractions could develop CSF-2, which was also sedimented by centrifugation. The specific activity of CSF-1 was increased in 20-30% AmS fractions, but was not enhanced further by NaF and/or ATP. CSF-2 could develop only in AmS fractions of fresh cytosols above 50% saturation which were devoid of CSF-1 and was reprecipitated from these fractions with AmS at 20-40% saturation with a 30 X increase in specific activity. CSF-2 development did not require ATP, but its rate increased with increasing temperature and was maximum around pH 5.5. These results show that CSF-1 and CSF-2 are separate entities and that CSF-2 is assembled from inactive precursors into an active, larger molecule.
Shibuya
E K
EK
Department of Zoology, University of Toronto, Ontario, Canada.
Masui
Y
Y
D000645:Ammonium Sulfate; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification*; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects*; D002458:Cell Fractionation / Q000379:methods; D002970:Cleavage Stage, Ovum / Q000166:cytology; D003600:Cytosol / Q000032:analysis; D010063:Ovum / Q000032:analysis*; D011894:Rana pipiens; D013997:Time Factors; D014461:Ultracentrifugation
D000970:Antineoplastic Agents; D000645:Ammonium Sulfate