Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1999-01-22
2000-12-26
Minnifield, Nita
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 6, 435 74, 435 79, 435193, 4352523, 4241411, 536 232, 536 2631, 536 2632, 536 235, 5303879, G01N 3353, G01N 33573, C12P 1318, A61K 39395, C07K 1600
Patent
active
06165731&
ABSTRACT:
A method is provided for identifying an compound that affects an activity of a polypeptide subunit of a SCF complex. The method includes contacting a sample comprising a chimeric SCF complex assembled from subunits derived from Saccharomyces cerevisiae or human and another species and a CDC34p polypeptide with the compound under conditions that allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a SCF substrate. The ubiquitination of the SCF substrate is measured. A chimeric in vitro assay system is provided for measuring CDC53p or CUL1p activity, comprising a CDC4p, CDC34p, and a SKP1p polypeptide, and either a CDC53p or CUL1p polypeptide. In this assay the CDC4p, CDC34p, and SKP1p polypeptide are either a yeast polypeptide or a polypeptide from another species, and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a yeast polypeptide and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a polypeptide from another species. A method is further provided for identifying a compound that affects the ability of a CDC4p, a SKP1p, a CDC34p, and a CDC53p or a CUL1p to ubiquitinate a substrate. The method includes contacting a sample comprising a CDC4p, a SKP1p, a CDC34p, and a CDC53p or CUL1p, with the compound under conditions sufficient to allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a substrate for ubiquitination. The ability of the CDC4p, the SKP1p, the CDC34p, and the CDC53p or CUL1p, to ubiquitinate the substrate is measured. A method is also provided of identifying a polypeptide having a function of a CDC4 subunit of SCF. A method is further provided for identifying a polypeptide as a substrate for a ubiquitination reaction.
REFERENCES:
Renny Feldman et al; A Complex of CDC4P, SKP1PAND CDC53P/Cullin Catalyzes Ubiquitination of the Phosphorylated CDK Inhibitor SIC1P; Cell, 91; 221-230, 1997.
Correll Craig C.
Deshaies Raymond
Lyapina Svetlana
Baskar Padma
California Institute of Technology
Haile Lisa A.
Minnifield Nita
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