Abstract
To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). The spindle checkpoint protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Animals
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Calcium-Binding Proteins / analysis*
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Calcium-Binding Proteins / chemistry
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Calcium-Binding Proteins / isolation & purification*
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Cell Cycle Proteins / analysis*
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / isolation & purification*
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Cloning, Molecular / methods
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / isolation & purification*
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Escherichia coli
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Humans
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Mad2 Proteins
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Magnetic Resonance Spectroscopy
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Ovum
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Protein Structure, Tertiary
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Repressor Proteins / analysis*
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Repressor Proteins / chemistry
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Repressor Proteins / isolation & purification*
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Ubiquitin-Protein Ligase Complexes / antagonists & inhibitors*
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Xenopus laevis
Substances
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Calcium-Binding Proteins
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Cell Cycle Proteins
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Enzyme Inhibitors
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MAD2L1 protein, human
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Mad2 Proteins
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Repressor Proteins
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome