Uridine phosphorylase association with vimentin - Intracellular distribution and localization

Citation
Rl. Russell et al., Uridine phosphorylase association with vimentin - Intracellular distribution and localization, J BIOL CHEM, 276(16), 2001, pp. 13302-13307
Citations number
69
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
276
Issue
16
Year of publication
2001
Pages
13302 - 13307
Database
ISI
SICI code
0021-9258(20010420)276:16<13302:UPAWV->2.0.ZU;2-Q
Abstract
Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathw ay, is associated with the intermediate filament protein vimentin, in NIH 3 T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylas e inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimen tin copurification with UPase was confirmed using both Western blot analysi s and MALDI-MS methods. Separation of cytosolic proteins using gel filtrati on chromatography yields a high molecular weight complex containing UPase a nd vimentin, Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pM and a stoichiometry of 1:2. Im munofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtu bule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin, Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin, Appro ximately 60-70% of the total UPase exists in the cytosol as a soluble prote in. Sequential extraction of MH 3T3 or colon 26 cells liberates an addition al 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demons trate for the first time that UPase is associated with vimentin and the exi stence of an enzymatically active cytoskeleton-associated UPase.