Uridine phosphorylase association with vimentin. Intracellular distribution and localization.
Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and 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. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule 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. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.
Department of Internal Medicine, Section of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
R L
RL
Russell
D
D
Cao
D
D
Zhang
R E
RE
Handschumacher
G
G
Pizzorno
D016475:3T3 Cells; D000818:Animals; D002846:Chromatography, Affinity; D003106:Colon; D003599:Cytoskeleton / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014543:Uridine Phosphorylase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D014746:Vimentin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014746:Vimentin; D014543:Uridine Phosphorylase