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M-phase promoting factors from eggs of Xenopus laevis
Abstract:
When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arrested at the G2/prophase boundary, it induces rapid entry of the cells into M-phase. MPF is present in late G2 and in M-phase of a variety of cell types, such as Xenopus eggs (naturally arrested in M), cleaving embryos, yeast, HeLa, and CHO cultures. MPF has been purified approximately 50-fold from eggs. It is stabilized by gamma-thio-ATP and by phosphoprotein phosphatase inhibitors. It runs as a protein of approximately 100 kd size on gel filtration. Oocytes contain a precursor of MPF, which is activated by post-translational means when a small amount of purified MPF is injected into the cell. Thus, MPF appears to be an auto-activating cytoplasmic trigger of M-phase. At anaphase of the cell cycle, MPF is inactivated due to the appearance of an 'anti-MPF' activity. Monoclonal antibodies have been prepared to partially purified MPF stabilized by gamma-thio-ATP, and several preparations which inactivate MPF were obtained. The antibodies are directed against thio-phosphate groups carried by a set of proteins including MPF. This indicates that MPF is present in our active preparations as a thio-phosphoprotein. These and other data suggest that MPF is normally activated in the cell cycle by a phosphorylation reaction.
Insights
M-phase promoting factor (MPF) triggers cell cycle entry into M-phase. MPF activation involves phosphorylation and inactivation by anti-MPF activity, revealing its role as a cell cycle regulator.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- M-phase promoting factor (MPF) is crucial for cell cycle progression.
- Xenopus oocytes are naturally arrested at the G2/prophase boundary.
- MPF is present in various cell types during specific cell cycle phases.
Purpose of the Study:
- To investigate the role of MPF as a trigger for M-phase entry.
- To characterize the activation and inactivation mechanisms of MPF.
- To identify the molecular nature of MPF.
Main Methods:
- Injection of purified MPF into Xenopus oocytes.
- Purification of MPF from Xenopus eggs.
- Stabilization of MPF using gamma-thio-ATP and phosphatase inhibitors.
- Gel filtration for size determination.
- Production of monoclonal antibodies against MPF.
Main Results:
- MPF injection rapidly induces M-phase entry in oocytes.
- MPF is a ~100 kd protein, present as a thio-phosphoprotein.
- Oocytes contain an MPF precursor activated by post-translational modification.
- An 'anti-MPF' activity inactivates MPF at anaphase.
- Monoclonal antibodies confirm MPF is a thio-phosphoprotein.
Conclusions:
- MPF acts as an auto-activating cytoplasmic trigger for M-phase.
- MPF activation likely involves a phosphorylation reaction.
- MPF regulation is critical for cell cycle control, with inactivation by anti-MPF activity at anaphase.