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Molecular characteristics of cytostatic factors in amphibian egg cytosols
1Department of Zoology, University of Toronto, Ontario, Canada.
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.
Insights
Researchers identified two forms of cytostatic factor (CSF) in amphibian eggs: CSF-1 and CSF-2. CSF-1 is a smaller, unstable protein sensitive to proteases, while CSF-2 is a large, highly stable molecule with unique properties.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Amphibian zygotes injected with unactivated egg cytosol arrest at metaphase.
- This cell cycle arrest is mediated by a factor termed cytostatic factor (CSF).
- CSF activity is modulated by calcium ions, leading to distinct primary (CSF-1) and secondary (CSF-2) forms.
Purpose of the Study:
- To partially purify and characterize the molecular properties of CSF-1 and CSF-2.
- To investigate the stability and sensitivity of both CSF forms to various treatments.
Main Methods:
- Partial purification using ammonium sulfate precipitation, sucrose density gradient centrifugation, and gel filtration.
- Analysis of molecular characteristics via SDS-polyacrylamide gel electrophoresis (PAGE) and Sepharose column chromatography.
- Assessment of stability through treatment with proteases, RNAse, varying pH, and denaturing agents.
Main Results:
- CSF-1 is a protease-sensitive, RNAse-resistant protein with a sedimentation coefficient of 3S, unstable at 25°C.
- CSF-2 is an extremely large molecule (>2 x 10^6 Da), stable at high pH and salt concentrations, and resistant to RNAse.
- CSF-2 is degraded by specific proteases (trypsin, alpha-chymotrypsin, papain) but not S. aureus V8 protease; its activity is concentration-independent.
Conclusions:
- Distinct molecular properties differentiate CSF-1 and CSF-2, suggesting different roles or origins.
- CSF-2's large size and extreme stability indicate a unique molecular structure and function in cell cycle regulation.
- Further characterization of these CSF forms can elucidate mechanisms of cell cycle control in early development.