title: Molecular characteristics of cytostatic factors in amphibian egg cytosols.
abstract: In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated 'cytostatic factor, (CSF)'. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20-30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 x 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.
authors.affiliation: Department of Zoology, University of Toronto, Ontario, Canada.
authors.forename: E K
authors.initials: EK
authors.lastname: Shibuya
authors.forename: Y
authors.initials: Y
authors.lastname: Masui
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003600:Cytosol / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D010063:Ovum / Q000737:chemistry / Q000502:physiology*; D017231:Proto-Oncogene Proteins c-mos / Q000302:isolation & purification* / Q000494:pharmacology; D011894:Rana pipiens
chemical_list: D017231:Proto-Oncogene Proteins c-mos