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Uridine phosphorylase association with vimentin. Intracellular distribution and localization
1Department of Internal Medicine, Section of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
The Journal of Biological Chemistry
|March 30, 2001
Summary
Uridine phosphorylase (UPase) binds to vimentin, a key cytoskeletal protein. This association, confirmed in cell lines, reveals an enzymatically active, cytoskeleton-associated UPase for the first time.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Uridine phosphorylase (UPase) is crucial for pyrimidine salvage.
- The cellular localization and interactions of UPase are not fully understood.
Purpose of the Study:
- To investigate the association of UPase with cellular structures.
- To determine if UPase interacts with the intermediate filament protein vimentin.
Main Methods:
- Affinity chromatography using a UPase inhibitor.
- Western blot analysis and MALDI-MS for protein identification.
- Gel filtration chromatography, in vitro binding assays, and immunofluorescence microscopy.
Main Results:
- UPase was purified and found to copurify with vimentin.
- In vitro studies showed a high-affinity binding between recombinant UPase and vimentin.
- Immunofluorescence confirmed UPase association with vimentin in intact cells, persisting even after microtubule depolymerization.
Conclusions:
- UPase is associated with both soluble and insoluble vimentin pools.
- A significant portion of UPase activity is linked to the cytoskeleton.
- This study demonstrates, for the first time, an enzymatically active, cytoskeleton-associated UPase.